Methods of detecting and controlling mucoid Pseudomonas biofilm production

ABSTRACT

Compositions and methods for detecting and controlling the conversion to mucoidy in  Pseudomonas aeruginosa  are disclosed. The present invention provides for detecting the switch from nonmucoid to mucoid state of  P. aeruginosa  by measuring mucE expression or MucE protein levels. The interaction between MucE and AlgW controls the switch to mucoidy in wild type  P. aeruginosa . Also disclosed is an alginate biosynthesis heterologous expression system for use in screening candidate substances that inhibit conversion to mucoidy.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 11/730,186, filed on Mar. 29, 2007, now U.S. Pat. No. 7,781,166and claims the benefit of the filing date of U.S. Provisional PatentApplication No. 60/787,497, filed Mar. 31, 2006, which is incorporatedherein by reference in its entirety.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH AND DEVELOPMENT

Statement under MPEP 310. The U.S. government has a paid-up license inthis invention and the right in limited circumstances to require thepatent owner to license others on reasonable terms as provided for bythe terms of NNA04CC74G awarded by the National Aeronautics and SpaceAdministration (NASA).

Part of the work performed during development of this invention utilizedU.S. Government funds. The U.S. Government has certain rights in thisinvention.

NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT

Not Applicable.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the identification and use of positiveregulators of alginate production in Pseudomonas aeruginosa. One aspectof the invention provides compositions and methods for the earlydetection and diagnosis of the conversion to mucoidy of Pseudomonasaeruginosa. The present invention also provides a molecular mechanismfor detecting the conversion from the nonmucoid to the mucoid state,including molecular probes for the early detection of this diseasestate.

2. Background Art

Cystic Fibrosis (CF) is the most common inheritable lethal disease amongCaucasians. The leading cause of high morbidity and mortality in CFpatients are the chronic respiratory infections caused by Pseudomonasaeruginosa. Pseudomonas aeruginosa is an aerobic, motile, gram-negativebacterium with a simple metabolic demand that allows it to thrive indiverse environments. P. aeruginosa normally inhabits soil, water, andvegetation. Although it seldom causes disease in healthy people, P.aeruginosa is an opportunistic pathogen associated with fatal pneumoniain patients with CF, as well as patients with compromised immune systemsand chronic infections such as non-cystic fibrosis bronchiectasis andurinary tract infections.

In CF patients, the initially colonizing P. aeruginosa strains arenonmucoid but in the CF lung, after a variable period, often one or twoyears, they inevitably convert into the mucoid form. Mucoid strains ofP. aeruginosa grow as biofilms in the airways of CF patients (Yu, H.,and N. E. Head, Front Biosci. 7:D442-57 (2002)). Biofilms refer tosurface-attached bacterial communities encased in a glycocalyx matrix(Costerton, J. W., et al., Science 284:1318-22 (1999)). Mucoid P.aeruginosa biofilms are microcolonies embedded in a capsule composed ofcopious amounts of alginate, an exopolysaccharide (Govan, J. R., and V.Deretic, Microbiol. Rev. 60:539-74 (1996)) and are resistant to hostdefenses (Ramsey, D. M., and D. J. Wozniak, Mol. Microbiol. 56:309-22(2005)).

The emergence of mucoid strains of P. aeruginosa in CF lungs signals thebeginning of the chronic phase of infection and is associated withfurther disease deterioration and poor prognosis (Lyczak, J. B., et al.,Clin. Microbiol. Rev. 15:194-222 (2002)). The chronic phase of infectiondue to P. aeruginosa is characterized by pulmonary exacerbations (fever,elevated white blood cell count, increased sputum production, anddecreased pulmonary function) that require antimicrobial therapy(Miller, M. B., and Gilligan, P. H., J. Clin. Microbiol. 41:4009-4015(2003)). CF exacerbations are typically interspersed with interveningperiods of relative quiescence, with each phase lasting various lengthsof time (Miller, M. B., and Gilligan, P. H., J. Clin. Microbiol.41:4009-4015 (2003)). However, lung function continuously declines, theinfecting strains become increasingly resistant, and inevitably, thepatient succumbs to cardiopulmonary failure (Miller, M. B., andGilligan, P. H., J. Clin. Microbiol. 41:4009-4015 (2003)).

There is a growing consensus that the lung pathology that occurs duringchronic P. aeruginosa infection is due to a large extent to the immuneresponse directed against pseudomonal biofilms (Miller, M. B., andGilligan, P. H., J. Clin. Microbiol. 41:4009-4015 (2003)). High levelsof cytokines and leukocyte-derived proteases can be detected in airwayfluid from CF patients and are believed to be responsible for much ofthe lung damage that occurs in this patient population (Miller, M. B.,and Gilligan, P. H., J. Clin. Microbiol. 41:4009-4015 (2003)). Alginateappears to protect P. aeruginosa from the consequences of thisinflammatory response as it scavenges free radicals released byactivated macrophages (Simpson, J. A., et al., Free Rad. Biol. Med.6:347-353 (1989)). The alginate mucoid coating also leads to theinability of patients to clear the infection, even under aggressiveantibiotic therapies, most probably because it provides a physical andchemical barrier to the bacterium (Govan and Deretic, Microbiol. Rev.60:539-574 (1996)).

Early aggressive antibiotic treatment of the initial colonizingnon-mucoid P. aeruginosa population might prevent or at least delaychronic pulmonary infection. However, questions still remain as towhether such treatment should be performed routinely or only duringpulmonary exacerbation, and whether the regimen could potentially leadto the emergence of resistant strains (Ramsey and Wozniak, Mol.Microbiol. 56:309-322 (2005)). Since P. aeruginosa is inherentlyresistant to many antibiotics at concentrations that can be achieved invivo, with the exception of ciprofloxacin, those to which it issensitive need to be given intravenously (Wilson and Dowling, Thorax53:213-219 (1998)). However, long-term, aggressive antibiotic treatmentis not without side effects. Therefore, it would be more beneficial toplace the emphasis on aggressive treatment strategies before the in vivoswitch to mucoidy since once chronic infection is established, it israrely possible to eradicate it even with intensive, antibiotic therapy.Thus, early detection of conversion to mucoidy in patients is desired toallow aggressive therapy, thereby preventing further diseasedeterioration.

Synthesis of alginate and its regulation has been the object of numerousstudies (Govan, J. R., and V. Deretic, Microbiol. Rev. 60:539-74 (1996);Ramsey, D. M., and D. J. Wozniak, Mol. Microbiol. 56:309-22 (2005)).Alginate production is positively and negatively regulated in wild-typecells.

Three tightly linked genes algU, mucA, and mucB have been previouslyidentified with a chromosomal region shown by genetic means to representthe site where mutations cause conversion to mucoidy (see U.S. Pat. Nos.6,426,187, 6,083,691, 5,591,838, and 5,573,910, incorporated herein byreference in their entireties).

Positive regulation centers on the activation of the alginatebiosynthetic operon (Govan, J. R., and V. Deretic, Microbiol. Rev.60:539-74 (1996)). Positive regulators include the alternativestress-related sigma factor AlgU (Martin, D. W., et al., Proc. Natl.Acad. Sci. 90:8377-81 (1993)), also called AlgT (DeVries, C. A., and D.E. Ohman, J. Bacteriol. 176:6677-87 (1994)), and transcriptionalactivators AlgR and AlgB, which belong to a bacterial two componentsignaling system. The cognate kinase of AlgB is KinB (Ma, S., et al., J.Biol. Chem. 272:17952-60 (1997)) while AlgZ (Yu, H., et al., J.Bacteriol. 179:187-93 (1997)) may be the kinase that phosphorylatesAlgR. However, unlike a typical two-component system, alginateoverproduction is independent of phosphorylation of AlgR or AlgB (Ma,S., et al., J. Bacteriol. 180:956-68 (1998)).

Negative regulation of alginate has focused on the post-translationalcontrol of AlgU activity. In alginate regulation, the master regulatoris AlgU and the signal transducer is MucA, a trans-inner membraneprotein whose amino terminus interacts with AlgU to antagonize theactivity of AlgU, and the carboxyl terminus with MucB, another negativeregulator of alginate biosynthesis. The algUmucABC cluster is conservedamong many Gram-negative bacteria. AlgU belongs to the family ofextracytoplasmic function (ECF) sigma factors that regulate cellularfunctions in response to extreme stress stimuli. The action of ECF sigmafactors is negatively controlled by MucA, MucB and MucC. This set ofproteins forms a signal transduction system that senses and responds toenvelope stress.

MucA is the anti-sigma factor that binds AlgU and antagonizes itstranscriptional activator activity (Schurr, M. J., et al., J. Bacteriol.178:4997-5004 (1996)). Consequently, inactivation of mucA in P.aeruginosa strain PAO1 results in the mucoid phenotype (Alg+) (Martin,D. W., et al., Proc. Natl. Acad. Sci. USA 90:8377-81 (1993); Mathee, K.,et al., Microbiology 145:1349-57 (1999)). Clinical mucoid isolates of P.aeruginosa carry recessive mutations in mucA (Anthony, M., et al., J.Clin. Microbiol. 40:2772-8 (2002); Boucher, J. C., et al., Infect.Immun. 65:3838-46 (1997)). The transition from a non-mucoid to mucoidvariant occurs in concurrence with the mucA22 allele after exposure tohydrogen peroxide, an oxidant in neutrophils (Mathee, K., et al.,Microbiology 145:1349-57 (1999)).

MucB is located in the periplasm in association with the periplasmicportion of MucA (Mathee, K., et al., J. Bacteriol. 179:3711-20 (1997);Rowen, D. W., and V. Deretic, Mol. Microbiol. 36:314-27 (2000)). MucC isa mild negative regulator whose action is in synergy with MucA or MucB(Boucher, J. C., et al., Microbiology 143:3473-80 (1997)). MucD is anegative regulator whose dual functions include periplasmic serineprotease and chaperone activities that are thought to help removemisfolded proteins of the cell envelope for quality control (Boucher, J.C., et al., J. Bacteriol. 178:511-23 (1996); Yorgey, P., et al., Mol.Microbiol. 41:1063-76 (2001)).

Overproduction of alginate is an important virulence factor forbacterial biofilm formation in vivo. Alginate protects the bacteriumfrom oxidative stress by scavenging the reactive oxygen species (Learn,D. B., et al., Infect. Immun. 55:1813-8 (1987); Simpson, J. A., et al.,Free Radic. Biol. Med. 6:347-53 (1989)).

There is a significant and urgent need in hospitals and clinicallaboratories for a rapid, sensitive and accurate diagnostic test fordetection of potential conversion to mucoidy of P. aeruginosa prior tothe detection of the emergence of a mucoid colony morphology on a growthplate in a laboratory.

BRIEF SUMMARY OF THE INVENTION

The present invention describes the identification and use of mucE, apositive regulator of alginate production in P. aeruginosa. Induction ofmucE causes mucoid conversion in P. aeruginosa.

One object of this invention is to provide compositions for the earlydetection and diagnosis of the conversion to mucoidy of Pseudomonasaeruginosa. The present invention also provides molecular probes todetect the conversion from the nonmucoid to the mucoid state, viaNorthern blot, RT-PCR, or real-time RT-PCR, including diagnostic kitsfor the early detection of this disease state.

Another object of this invention is to provide methods for the earlydetection and diagnosis of the conversion to mucoidy of Pseudomonasaeruginosa. One method for detecting a cell converted to mucoidyinvolves obtaining a cell sample suspected of conversion to mucoidy,contacting messenger RNA from the cell sample with a mucE nucleic acidsegment, and detecting the presence of increased hybridized complexes,wherein the presence of increased hybridized complexes is indicative ofconversion to mucoidy. A six fold increase of mucE messenger RNA issufficient to cause conversion to mucoidy in mucA+ wild type cells.Thus, early detection of conversion to mucoidy is possible by detectingand measuring mucE expression as compared to the baseline expressionlevel of mucE in non-mucoid cells.

Early detection for the trend of increased expression of the mucEmessage in various samples, including the sputum samples from patientswith cystic fibrosis, samples from patients carrying endotracheal tubes,and urinary tract catheters would provide an indication that thecolonizing bacteria has started to enter the biofilm mode of growth,thereby requiring immediate administration of aggressive antibiotictherapy.

A further embodiment of this invention are the use of MucE antibodiesand methods of using MucE antibodies for detecting the conversion tomucoidy of P. aeruginosa.

A further embodiment of this invention is a method for preventing theconversion to mucoidy of P. aeruginosa by blocking mucE expression orMucE activity. Mucoid P. aeruginosa biofilms can be formed via twomeans: the mutations in mucA (see U.S. Pat. Nos. 6,426,187, 6,083,691,and 5,591,838), and increased expression of mucE. mucE acts upstream ofmucA, thus, the control of mucoidy mediated by mucE occurs before themucA mutation. Therefore, inhibition of MucE activity provides a meansto prevent conversion to mucoidy during the early stage of bacterialcolonization.

In still further embodiments, the present invention concerns a methodfor identifying new compounds that inhibit mucE gene expression or MucEfunction, which may be termed “candidate substances.” Such compounds mayinclude anti-sense oligonucleotides or molecules that block or repressthe mucE promoter, or molecules that directly bind to MucE to block theactivity of MucE.

The present invention also provides for a method for screening acandidate substance for preventing P. aeruginosa conversion to mucoidycomprising contacting E. coli bacteria with an effective amount of acandidate substance; and assaying for reporter gene activity, wherein adecrease in the expression of the reporter gene indicates inhibition ofmucE promoter activity.

Another object of the present invention is AlgW, a positive regulatorfor alginate production, and the use of AlgW as a potential drug target.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 shows the nucleotide sequence of mucE in P. aeruginosa (SEQ IDNO:1). The mucE gene is an unclassified ORF (PA4033) in the genomes ofPAO1 and PA14. It encodes a small peptide of 89 amino acids with amolecular mass of 9.5 kDa.

FIG. 2 shows the amino acid sequence of MucE in P. aeruginosa (SEQ IDNO:2). MucE has a predicted N-terminal leader peptide of 36 amino acids,which is likely to direct the native peptide of MucE to the innermembrane for processing and export to the periplasm or outer membrane ofP. aeruginosa. The WVF at the C-terminus is the signal for alginateinduction.

FIG. 3 shows the nucleotide sequence of the homolog of mucE in P.fluorescence Pf-5 (SEQ ID NO:3).

FIG. 4 shows the amino acid sequence of the homolog of MucE in P.fluorescence Pf-5 (SEQ ID NO:4).

FIG. 5 shows the number of mariner transposon insertions per TA site inthe algU promoter region of four strains of P. aeruginosa. FIG. 5A showsthe frequency of the insertions in each P. aeruginosa strain. FIG. 5Bshows the sequence of the algU promoter region (SEQ ID NO: 16)containing all TA sites with an assigned number matching to FIG. 5A.

FIG. 6 shows the levels of alginate, AlgU and MucB in P. aeruginosamucoid mutants caused by induction of algUmucABC in comparison with thewild type PAO1 (B). FIG. 6A shows the amounts of alginate (μgalginate/mg protein) that were measured for 4-72 h. Asterisk indicatessignificant differences at P<0.05 in comparison with the same time pointin PAO1. FIG. 6B is a Western blot analysis of the total proteinextracts from the same cells as above were probed by anti-AlgU (Schorr,M. J., et al., J. Bacteriol. 178:4997-5004 (1996)) and anti-MucB(Boucher, J. C., et al., J. Bacteriol. 178:511-23 (1996)) monoclonalantibodies. The genotype of each mutant is shown. The number below eachblot was the ratio of internally normalized protein relative to thelevel of PAO1 at the same time point. The +oe superscript used in FIG.6A refers to the overexpression of the algU mucABC operon.

FIG. 7 shows the levels of alginate, the expression of AlgU and MucB inVE2 (PAO1 mucE^(+oe)) as detected by Western blots (FIG. 7A) and RT-PCR(FIG. 7B). Bacterial cells were grown under the same conditions asdescribed in Methods, and were subjected to the same treatments as inFIG. 6. Asterisk in alginate production indicates significantdifferences compared with PAO1 at the same time point as in FIG. 6. Theratio of internally normalized AlgU and MucB to those of PAO1 isshown.—in FIG. 7B indicates the RT minus controls.

FIG. 8 shows upregulation of AlgU in VE13 (PAO1 kinB⁻) (FIG. 8A) inassociation with increased alginate production. FIG. 8B: Western blotsshowing the levels of AlgU and MucB in various mutants after 24 hgrowth. FRD2 carries the algT18 suppressor mutant while FRD2-VE1 is likeVE1 with the insertion in the algU promoter. VE3-NM1 to -NM4 are thespontaneous nonmucoid mutants with suppressors inactivating algU.VE3NM3+algU: pUCP20-algU in trans. VE22: cupB5^(+oe) and VE24: oprL⁺ butwith reduced expression of oprL due to production of the antisense RNA.

FIG. 9 shows the regulatory cascade of alginate production in P.aeruginosa. AlgU is the alginate-specific sigma factor, whose activityis antagonized by anti-sigma factor, MucA. MucA is an inner membraneprotein with its C-terminus in the periplasm, and its N-terminusinteracting with AlgU in cytoplasm. The alginate operon consists of 12genes encoding biosynthetic enzymes, thus collectively termed “alginateengine.” The enzymes AlgI, AlgJ, and AlgF are involved in O-acetylationof alginate. AlgK is needed for formation of the alginate polymer andAlgE for the export of alginate across the membrane.

FIG. 10 is a map of the expression vector pUCP20-Gm-mucE. The expressionvector contains the coding region of the mucE gene driven by a promoterderived from the gentamicin (Gm) cassette of pFAC. This promoter ishighly expressive in P. aeruginosa. This construct can render thenonmucoid PAO1 mucoid while the control backbone vector without mucE hasno effect on the phenotype.

FIG. 11 shows an alignment of the mucE homologs identified from thecompleted and partially completed genomes of three species within thegenus of Pseudomonas. The three species are PA: Pseudomonas aeruginosa;PF: Pseudomonas fluorescens; and PS: Pseudomonas syringae. The strainsshown are: PA-PAO1 (SEQ ID NO: 23), Pseudomonas aeruginosa PAO1 (causesopportunistic infections in humans); PA-PA14 (SEQ ID NO: 22),Pseudomonas aeruginosa UCBPP PA14 (human clinical isolate); PA-2192 (SEQID NO: 20), Pseudomonas aeruginosa 2192 (CF patient isolate); PA-C3719(SEQ ID NO: 21), Pseudomonas aeruginosa C3719 (unknown source butprobably clinical origin); PS-PPH (SEQ ID NO: 26), Pseudomonas syringaepv. phaseolicola 1448A (causes halo blight on beans); PS-PTO (SEQ ID NO:27), Pseudomonas syringae pv. tomato DC3000 (bacterial speck disease ontomato plants); PS-SB728 (SEQ ID NO: 28), Pseudomonas syringae pv.syringae B728a (brown spot disease on beans); PF-PF5 (SEQ ID NO: 24),Pseudomonas fluorescens Pf-5 (Saprophyte) (the production of a number ofantibiotics as well as the production of siderophores by this strain caninhibit phytopathogen growth); and PF-PFO1 (SEQ ID NO: 25), Pseudomonasfluorescens PfO-1 (microorganism of putrefaction and well adapted tosoil environments).

FIG. 12 shows an alignment of the algal homologs identified from thecompleted and partially completed genomes of three species within thegenus of Pseudomonas. The three species are PA: Pseudomonas aeruginosa;PF: Pseudomonas fluorescens; and PS: Pseudomonas syringae. All thesespecies have the capacity to overproduce alginate. The strains shown arethe same as for FIG. 11, and include AlgW homologs for PA-2192 (SEQ IDNO: 29), PA-C3719 (SEQ ID NO: 30), PA-PA14 (SEQ ID NO: 31), PA-PAO1 (SEQID NO: 32), PF-PF5 (SEQ ID NO: 33), PF-PFO1 (SEQ ID NO: 34), PS-PPH (SEQID NO: 35), PS-PTO (SEQ ID NO: 36), and PS-SB728 (SEQ ID NO: 37). Thepredicted functional domains of AlgW include an N-terminal signalpeptide sequence at amino acids 1-27, a trypsin domain (peptidaseactivity, serine at AlgW 227 is conserved) at amino acids 114-260, and aPDZ domain at amino acids 270-380.

FIG. 13 shows the detection of N-terminal His-tag labeled MucE proteinvia Western Blot with anti-penta-his monoclonal antibody and SDS-PAGEwith Coomassie blue.

FIG. 14 shows the sequence of mucE (SEQ ID NO: 2; amino acid sequence ofmucE) and the phenotypes of the different translational mucE-phoAfusions (SEQ ID NO: 17; nucleic acid sequence of the full-lengthmucE-phoA fusion). The location of the mariner transposon bearing theaacC1 gene conferring Gm^(r) in the chromosome of the mucoid mutantsPAO1VE2 and PA14DR4 is shown. Different lengths of mucE sequences werefused with phoA without the leader signal peptide sequence todemonstrate the effect of the signal sequence on translocation acrossthe inner membrane to the periplasm. 1. Negative control, no 5′ leaderpeptide sequence (no sig phoA); 2. Positive control, the wild-type E.coli phoA leader sequence restored in the construct by directly fusingit with phoA (Ec wt-phoA); 3. Full-length mucE-phoA; 4. mucE with thepredicted N-terminal leader sequence fused with phoA; 5. partial mucEN-terminal leader sequence fused with phoA; 6. C-terminal mucE with ATGfused with phoA. The exact phoA fusion sites are as indicated in themucE sequence. The leader sequence of mucE with max cleavage site isbetween pos. 36 (P) and 37 (A) (box).

FIG. 15 shows an alignment of MucP (SEQ ID NO: 19) and the Escherichiacoli orthologue RseP (SEQ ID NO: 18). Identical amino acids are markedby an asterisk (*). The two terminal protease domains are shown in lightgray and the two PDZ domains are shown in medium gray. The overlappingregion containing both a portion of the protease domain and a portion ofthe PDZ domain is shown in dark gray.

DETAILED DESCRIPTION OF THE INVENTION

Infections due to P. aeruginosa are recognized by the medical communityas particularly difficult to treat. In particular, the emergence of amucoid phenotype of P. aeruginosa in CF lungs is associated with furtherdisease deterioration and poor prognosis. A patient's prognosis forrecovery from an infection caused by mucoid P. aeruginosa is enhancedwhen the diagnosis is made and appropriate treatment initiated as earlyin the course of infection as possible before the number of bacteria inthe host becomes overwhelming and much more difficult to bring undercontrol. Thus, early detection of P. aeruginosa conversion to mucoidy inpatients is particularly desired to allow aggressive therapy, therebypreventing further disease deterioration.

The present application describes the identification of a positiveregulator involved in alginate and biofilm production in P. aeruginosa,termed mucE (SEQ ID NOs:1-2) (GenBank accession numbers DQ352561 (PAO1mucE) and DQ352562 (PA14 mucE)). Induction of mucE causes mucoidconversion in P. aeruginosa.

One object of this invention is to provide compositions for the earlydetection and diagnosis of the conversion to mucoidy of Pseudomonasaeruginosa in biological specimens. By “early detection” is meantdetecting P. aeruginosa conversion to mucoidy using certain assaymethods, including but not limited to, methods involving the use of anucleic acid probe or antibodies, 1 to 14 days, specifically 1 to 10days, more specifically 1 to 7 days, and most specifically 6 days, 5days, 4 days, 3 days, 2 days, 24 hours, 18 hours, 12 hours or 8 hoursbefore detecting the emergence of a mucoid colony morphology on a growthplate in a laboratory.

The present invention also provides molecular probes to detect theconversion from the nonmucoid to the mucoid state, including viaNorthern blot, RT-PCR, or real-time RT-PCR, including diagnostic kitsfor the early detection of this disease state.

The present invention is also directed to algW and the use of AlgW as apotential drug target. Contrary to previous findings, AlgW is a positiveregulator for alginate production. The algW gene and AlgW protein, thealgW homologs, and the uses thereof as described above for the P.aeruginosa mucE gene and MucE protein are also part of the presentinvention.

Another object of this invention are mucA mucoid mutants and the use ofthese mutants to screen for suppressors and potential toxin genes.Mucoid mutants with mucA mutations (see U.S. Pat. Nos. 6,426,187,6,083,691, and 5,591,838) have been previously detected from clinicalspecimens. The presence of these mutations is a poor prognosticator andrepresents the onset of chronic infection. Since the elevation of mucEcan cause the emergence of mucoid P. aeruginosa before mucA mutationsoccur, the involvement of mucE in alginate induction is upstream ofmucA.

Another object of this invention is to provide methods for the earlydetection and diagnosis of the conversion to mucoidy of Pseudomonasaeruginosa. One method for detecting a cell converted to mucoidyinvolves obtaining a biological specimen suspected of conversion tomucoidy, contacting messenger RNA from the specimen with a mucE nucleicacid segment, and detecting the presence of increased hybridizedcomplexes, wherein the presence of increased hybridized complexes overbaseline is indicative of conversion to mucoidy.

The biological specimen to be assayed for the presence of mucoid P.aeruginosa can be prepared in a variety of ways, depending on the sourceof the specimen. The specimen may be obtained from the following:patients with debilitated immune systems, sputum samples from patientswith pneumonia, endotracheal samples from intubating patients underintensive care, samples from urinary catheters, samples from wounds, andespecially from patients suffering from cystic fibrosis. Specimens maybe a sample of human blood, sputum, wound exudate, endotracheal samples,respiratory secretions, human tissues (e.g., lung) or a laboratoryculture thereof, and urine. Since alginate induction is synonymous withbiofilm formation in vivo, the increased expression of mucE may also beused to monitor the biofilm formation in a confined environment duringspace travel (astronauts).

A further embodiment of this invention is the use of MucE antibodies andmethods of using MucE antibodies for detecting the conversion to mucoidyof P. aeruginosa via ELISA or other immunoassays.

A further embodiment of this invention is a method for preventing theconversion to mucoidy of P. aeruginosa. In particular, the presentinvention concerns methods for identifying new compounds that inhibitmucE gene expression or MucE function, which may be termed “candidatesubstances.” Such compounds may include anti-sense oligonucleotides ormolecules that block or repress the mucE promoter.

Specifically, when the last three amino acids of MucE are changed fromWVF to other combinations, the majority of altered signals areineffective to induce mucoid biofilm production, indicating thespecificity of this signal in mucoid conversion. Thus, WVF is animportant signal for mucoid biofilm formation in P. aeruginosa. This WVFsignal plays a role in the bacterium's ability to overproduce alginateand enter a biofilm mode of growth via regulated proteolysis as depictedin FIG. 9. The present invention provides for methods to employ thesignal as a drug target. Diagnostic kits to screen for the presence ofthe signal in patients with chronic P. aeruginosa infections arecontemplated. In addition, methods to screen for compounds that inhibitthe function of this signal are also contemplated. Such compounds willhave a specific anti-biofilm function.

The present invention also provides for a method for screening acandidate substance for preventing P. aeruginosa conversion to mucoidycomprising contacting E. coli bacteria with an effective amount of acandidate substance; and assaying for reporter gene activity, wherein adecrease in the expression of the reporter gene indicates inhibition ofmucE promoter activity.

MucE homologs from other Pseudomonas species or strains are alsocontemplated (see FIG. 11). These Pseudomonas species and strainsinclude PA-PAO1, Pseudomonas aeruginosa PAO1 (causes opportunisticinfections in humans); PA-PA14, Pseudomonas aeruginosa UCBPP PA14 (humanclinical isolate); PA-2192, Pseudomonas aeruginosa 2192 (CF patientisolate); PA-C3719, Pseudomonas aeruginosa C3719 (unknown source butprobably clinical origin); PS-PPH, Pseudomonas syringae pv. phaseolicola1448A (causes halo blight on beans); PS-PTO, Pseudomonas syringae pv.tomato DC3000 (bacterial speck disease on tomato plants); PS-SB728,Pseudomonas syringae pv. syringae B728a (brown spot disease on beans);PF-PF5, Pseudomonas fluorescens Pf-5 (Saprophyte) (the production of anumber of antibiotics as well as the production of siderophores by thisstrain can inhibit phytopathogen growth); and PF-PFO1, Pseudomonasfluorescens PfO-1 (microorganism of putrefaction and well adapted tosoil environments). The mucE homologs and the use thereof as describedabove for the P. aeruginosa mucE gene and MucE protein are also part ofthe present invention.

Isolated polynucleotides comprising fragments containing one or moremucE consensus regions are also contemplated. The consensus regions areshown in FIG. 11.

By “isolated” polynucleotide is intended a nucleic acid molecule, DNA orRNA, circular or linear, which has been removed from its nativeenvironment. For example, recombinant DNA molecules contained in avector are considered isolated for the purposes of the presentinvention. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.

The term “positive regulator” as used herein, means that the inductionof expression and/or activity of such a gene encoding a functionalprotein causes alginate overproduction. Examples of positive regulatorsinclude algU, mucE, and algal.

The term “negative regulator” as used herein, means that the absence ofsuch a gene encoding a functional protein causes alginateoverproduction. Examples of negative regulators include kinB, mucA,mucB, and mucD.

The term “recombinant,” as used herein, means that a protein is derivedfrom recombinant (e.g., microbial) expression systems. The term“microbial” refers to recombinant proteins made in bacterial or fungal.(e.g., yeast) expression systems. As a product, the term “recombinantmicrobial” defines a protein produced in a microbial expression systemwhich is essentially free of native endogenous substances. Proteinexpressed in most bacterial cultures, e.g., E. coli, will be free ofglycan.

The term “DNA sequence” refers to a DNA polymer, in the form of aseparate fragment or as a component of a larger DNA construct.Preferably, the DNA sequences are in a quantity or concentrationenabling identification, manipulation, and recovery of the sequence andits component nucleotide sequences by standard biochemical methods, forexample, using a cloning vector. Such sequences are preferably providedin the form of an open reading frame uninterrupted by internalnontranslated sequences. Genomic DNA containing the relevant sequencescould also be used. Sequences of non-translated DNA may be present 5′ or3′ from the open reading frame, where the same do not interfere withmanipulation or expression of the coding regions.

The term “nucleotide sequence” refers to a heteropolymer ofdeoxyribonucleotides. DNA sequences encoding the proteins of thisinvention can be assembled from fragments and short oligonucleotidelinkers, or from a series of oligonucleotides, to provide a syntheticgene which is capable of being expressed in a recombinanttranscriptional unit.

The term “recombinant expression vector” refers to a replicable DNAconstruct used either to amplify or to express DNA which encodes therecombinant proteins of the present invention and which includes atranscriptional unit comprising an assembly of (1) a genetic element orelements having a regulatory role in gene expression, for example,promoters or enhancers, (2) a structure or coding sequence which istranscribed into mRNA and translated into protein, and (3) appropriatetranscription and translation initiation and termination sequences.Structural elements intended for use in yeast expression systemspreferably include a leader sequence enabling extracellular secretion oftranslated protein by a host cell. Alternatively, where recombinantprotein is expressed without a leader or transport sequence, it mayinclude an N-terminal methionine residue. This residue may optionally bysubsequently cleaved from the expressed recombinant protein to provide afinal product.

As used herein, the term “expression vector” refers to a construct madeup of genetic material (i.e., nucleic acids). Typically, a expressionvector contains an origin of replication which is functional inbacterial host cells, e.g., Escherichia coli, and selectable markers fordetecting bacterial host cells comprising the expression vector.Expression vectors of the present invention contain a promoter sequenceand include genetic elements as described herein arranged such that aninserted coding sequence can be transcribed and translated inprokaryotes or eukaroytes. In certain embodiments described herein, anexpression vector is a closed circular DNA molecule.

The term “expression” refers to the biological production of a productencoded by a coding sequence. In most cases, a DNA sequence, includingthe coding sequence, is transcribed to form a messenger-RNA (mRNA). Themessenger-RNA is then translated to form a polypeptide product which hasa relevant biological activity. Also, the process of expression mayinvolve further processing steps to the RNA product of transcription,such as splicing to remove introns, and/or post-translational processingof a polypeptide product.

The term “recombinant microbial expression system” means a substantiallyhomogeneous monoculture of suitable host microorganisms, for example,bacteria such as E. coli or yeast such as S. cerevisiae, which havestably integrated a recombinant transcriptional unit into chromosomalDNA or carry the recombinant transcriptional unit as a component of aresident plasmid. Generally, cells constituting the system are theprogeny of a single ancestral transformant. Recombinant expressionsystems as defined herein will express heterologous protein uponinduction of the regulatory elements linked to the DNA sequence orsynthetic gene to be expressed.

One embodiment of the present invention is a method of detectingconversion to mucoidy in Pseudomonas aeruginosa in a biological specimencomprising detecting MucE expression. A preferred embodiment is a methodof detecting conversion to mucoidy in Pseudomonas aeruginosa having anactive mucE gene product comprising the detection of the mucE messengerRNA in a sample suspected of conversion to mucoidy. In this case, thesequence encodes an active gene product and the sequence is detected byhybridization with a complementary oligonucleotide, to form hybridizedcomplexes. The presence of increased hybridized complexes is indicativeof conversion to mucoidy in Pseudomonas aeruginosa. The complementaryoligonucleotides may be 5′-TCAAAACACCCAGCGCAACTCGTCACG-3′, (SEQ ID NO:5)5′-AGTAGCGAAGGACGGGCTGGCGGT-3′, (SEQ ID NO:6) or5′-TTGGCTAACTGGCCGGAAACCCAT-3′ (SEQ ID NO:7).

A further embodiment of the present invention is the use of MucEantibodies and methods of using MucE antibodies for detecting theconversion to mucoidy of P. aeruginosa or for inhibiting MucE function.

In still further embodiments, the present invention concerns a methodfor identifying new compounds that inhibit transcription from the mucEpromoter, which may be termed as “candidate substances.” Such compoundsmay include anti-sense oligonucleotides or molecules that encouragerepression of the mucE promoter. The present invention provides for amethod for screening a candidate substance for preventing P. aeruginosaconversion to mucoidy comprising: contacting E. coli bacteria with aneffective amount of a candidate substance; and assaying for reportergene activity, wherein a decrease in the expression of the reporter geneindicates inhibition of MucE promoter activity.

In additional embodiments, the present invention also concerns a methodfor detecting mucoid Pseudomonas aeruginosa bacterium in a biologicalsample. The method comprises reacting a sample suspected of containingP. aeruginosa with a detergent, EDTA, and a monoclonal antibody orfragment thereof capable of specifically binding to MucE expressed by P.aeruginosa, separating the sample from unbound monoclonal antibody; anddetecting the presence or absence of immune complexes formed between themonoclonal antibody and MucE.

Polynucleotides

The DNA sequences disclosed herein will also find utility as probes orprimers in nucleic acid hybridization embodiments. Nucleotide sequencesof between about 10 nucleotides to about 20 or to about 30 nucleotides,complementary to SEQ ID NOs:1-4, will find particular utility, with evenlonger sequences, e.g., 40, 50, 100, even up to full length, being morepreferred for certain embodiments. The ability of such nucleic acidprobes to specifically hybridize to mucE-encoding sequences will enablethem to be of use in a variety of embodiments. For example, the probescan be used in a variety of assays for detecting the presence ofcomplementary sequences in a given sample. However, other uses areenvisioned, including the use of the sequence information for thepreparation of mutant species primers, or primers for use in preparingother genetic constructions.

Nucleic acid molecules having stretches of 10, 15, 20, 30, 50, or evenof 100 nucleotides or so, complementary to SEQ ID NOs:1 and 3, will haveutility as hybridization probes. These probes will be useful in avariety of hybridization embodiments, such as Southern and Northernblotting in connection with analyzing the complex interaction ofstructural and regulatory genes in diverse microorganisms and inclinical isolates from patients, including CF patients. The total sizeof fragment, as well as the size of the complementary stretch(es), willultimately depend on the intended use or application of the particularnucleic acid segment. Smaller fragments will generally find use inhybridization embodiments, wherein the length of the complementaryregion may be varied, such as between about 10 and about 100nucleotides, according to the complementary sequences one wishes todetect.

The use of a hybridization probe of about 10 nucleotides in lengthallows the formation of a duplex molecule that is both stable andselective. Molecules having complementary sequences over stretchesgreater than 10 bases in length are generally preferred, though, inorder to increase stability and selectivity of the hybrid, and therebyimprove the quality and degree of specific hybrid molecules obtained.One will generally prefer to design nucleic acid molecules havinggene-complementary stretches of 15 to 20 nucleotides, or even longerwhere desired. Such fragments may be readily prepared by, for example,directly synthesizing the fragment by chemical means, by application ofnucleic acid reproduction technology, such as the Polymerase ChainReaction (PCR) technology of U.S. Pat. No. 4,603,102 (hereinincorporated by reference) or by introducing selected sequences intorecombinant vectors for recombinant production.

Accordingly, the nucleotide sequences of the invention may be used fortheir ability to selectively form duplex molecules with complementarystretches of homologous, or heterologous genes or cDNAs. Depending onthe application envisioned, one will desire to employ varying conditionsof hybridization to achieve varying degrees of selectivity of probetowards target sequence. For applications requiring high selectivity,one will typically desire to employ relatively stringent conditions toform the hybrids, e.g., one will select relatively low salt and/or hightemperature conditions, such as provided by 0.02 M-0.15 M NaCl attemperatures of 50° C. to 70° C. Such selective conditions toleratelittle, if any, mismatch between the probe and the template or targetstrand, and would be particularly suitable for isolating functionallyrelated genes.

In certain instances, for example, where one desires to prepare mutantsemploying a mutant primer strand hybridized to an underlying template orwhere one seeks to isolate specific mutant mucE-encoding sequences fromrelated species, functional equivalents, or the like, less stringenthybridization conditions will typically be needed in order to allowformation of the heteroduplex. In these circumstances, one may desire toemploy conditions such as 0.15 M-0.9 M salt, at temperatures rangingfrom 20° C. to 55° C. Cross-hybridizing species can thereby be readilyidentified as positively hybridizing signals with respect to controlhybridizations. In any case, it is generally appreciated that conditionscan be rendered more stringent by the addition of increasing amounts offormamide, which serves to destabilize the hybrid duplex in the samemanner as increased temperature. Thus, hybridization conditions can bereadily manipulated, and thus will generally be a method of choicedepending on the desired results.

In certain embodiments, it will be advantageous to employ nucleic acidsequences of the present invention in combination with an appropriatemeans, such as a label, for determining hybridization. A wide variety ofappropriate indicator means are known in the art, including fluorescent,radioactive, enzymatic, biotinylated, and chemiluminescent labels, whichare capable of giving a detectable signal. Fluorophores, luminescentcompounds, radioisotopes and particles can also be employed. Inpreferred embodiments, one will likely desire to employ a fluorescentlabel or an enzyme tag, such as urease, alkaline phosphatase orperoxidase, instead of radioactive or other environmental undesirablereagents. In the case of enzyme tags, calorimetric indicator substratesare known which can be employed to provide a means visible to the humaneye or spectrophotometrically, to identify specific hybridization withcomplementary nucleic acid-containing samples.

In general, it is envisioned that the hybridization probes describedherein will be useful both as reagents in solution hybridization as wellas in embodiments employing a solid phase. In embodiments involving asolid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to aselected matrix or surface. This fixed, single-stranded nucleic acid isthen subjected to specific hybridization with selected probes underdesired conditions. The selected conditions will depend on theparticular circumstances based on the particular criteria required(depending, for example, on the G+C contents, type of target nucleicacid, source of nucleic acid, size of hybridization probe, etc.).Following washing of the hybridized surface so as to removenonspecifically bound probe molecules, specific hybridization isdetected, or even quantified, by means of the label.

Longer DNA segments will often find particular utility in therecombinant production of peptides or proteins. DNA segments whichencode peptide antigens from about 15 to about 50 amino acids in length,or more preferably, from about 15 to about 30 amino acids in length arecontemplated to be particularly useful. DNA segments encoding peptideswill generally have a minimum coding length in the order of about 45 toabout 150, or to about 90 nucleotides.

The nucleic acid segments of the present invention, regardless of thelength of the coding sequence itself, may be combined with other DNAsequences, such as promoters, additional restriction enzyme sites,multiple cloning sites, other coding segments, and the like, such thattheir overall length may vary considerably. It is contemplated that anucleic acid fragment of almost any length may be employed, with thetotal length preferably being limited by the ease of preparation and usein the intended recombinant DNA protocol. For example, nucleic acidfragments may be prepared in accordance with the present invention whichare up to 10,000 base pairs in length, with segments of 5,000, 3,000,2,000 or 1,000 base pairs being preferred and segments of about 500 basepairs in length being particularly preferred.

It will be understood that this invention is not limited to theparticular nucleic acid and amino acid sequences of SEQ ID NOs:1 and 3.Therefore, DNA segments prepared in accordance with the presentinvention may also encode biologically functional equivalent proteins orpeptides which have variant amino acids sequences. Such sequences mayarise as a consequence of codon redundancy and functional equivalencywhich are known to occur naturally within nucleic acid sequences and theproteins thus encoded. Alternatively, functionally equivalent proteinsor peptides may be created via the application of recombinant DNAtechnology, in which changes in the protein structure may be engineered,based on considerations of the properties of the amino acids beingexchanged.

Further embodiments of the invention include vectors comprisingpolynucleotides, which comprise a nucleotide sequence at least 95%identical, and more preferably at least 96%, 97%, 98% or 99% identical,to any of the nucleotide sequences of the vectors comprisingpolynucleotides described above.

Other embodiments of the invention include polynucleotides, whichcomprise a nucleotide sequence at least 95% identical, and morepreferably at least 96%, 97%, 98% or 99% identical, to any of thenucleotide sequences of the polynucleotides described above.

As a practical matter, whether any particular vector or polynucleotideis at least 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequenceaccording to the present invention, can be determined conventionallyusing known computer programs such as the Bestfit program (WisconsinSequence Analysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park 575 Science Drive, Madison, Wis. 53711).Bestfit uses the local homology algorithm of Smith and Waterman,Advances in Applied Mathematics 2:482-489 (1981), to find the bestsegment of homology between two sequences. When using Bestfit or anyother sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set, of course,such that the percentage of identity is calculated over the full lengthof the reference nucleotide sequence and that gaps in homology of up to5% of the total number of nucleotides in the reference sequence areallowed.

Codon Optimization

As used herein, the term “codon optimization” is defined as modifying anucleic acid sequence for enhanced expression in the cells of thevertebrate of interest, e.g., human, by replacing at least one, morethan one, or a significant number, of codons of the native sequence withcodons that are more frequently or most frequently used in the genes ofthat vertebrate. Various species exhibit particular bias for certaincodons of a particular amino acid.

In one aspect, the present invention relates to polynucleotideexpression constructs or vectors, and host cells comprising nucleic acidfragments of codon-optimized coding regions which encode therapeuticpolypeptides, and fragments, variants, or derivatives thereof, andvarious methods of using the polynucleotide expression constructs,vectors, host cells to treat or prevent disease in a vertebrate.

As used herein the term “codon-optimized coding region” means a nucleicacid coding region that has been adapted for expression in the cells ofa given vertebrate by replacing at least one, or more than one, or asignificant number, of codons with one or more codons that are morefrequently used in the genes of that vertebrate.

Deviations in the nucleotide sequence that comprise the codons encodingthe amino acids of any polypeptide chain allow for variations in thesequence coding for the gene. Since each codon consists of threenucleotides, and the nucleotides comprising DNA are restricted to fourspecific bases, there are 64 possible combinations of nucleotides, 61 ofwhich encode amino acids (the remaining three codons encode signalsending translation). Many amino acids are designated by more than onecodon. For example, the amino acids alanine and proline are coded for byfour triplets, serine and arginine by six, whereas tryptophan andmethionine are coded by just one triplet. This degeneracy allows for DNAbase composition to vary over a wide range without altering the aminoacid sequence of the proteins encoded by the DNA.

Consensus Sequences

The present invention is further directed to expression plasmids thatcontain chimeric genes which express therapeutic fusion proteins withspecific consensus sequences, and fragments, derivatives and variantsthereof. A “consensus sequence” is, e.g., an idealized sequence thatrepresents the amino acids most often present at each position of two ormore sequences which have been compared to each other. A consensussequence is a theoretical representative amino acid sequence in whicheach amino acid is the one which occurs most frequently at that site inthe different sequences which occur in nature. The term also refers toan actual sequence which approximates the theoretical consensus. Aconsensus sequence can be derived from sequences which have, e.g.,shared functional or structural purposes. It can be defined by aligningas many known examples of a particular structural or functional domainas possible to maximize the homology. A sequence is generally acceptedas a consensus when each particular amino acid is reasonably predominantat its position, and most of the sequences which form the basis of thecomparison are related to the consensus by rather few substitutions,e.g., from 0 to about 100 substitutions. In general, the wild-typecomparison sequences are at least about 50%, 75%, 80%, 90%, 95%, 96%,97%, 98% or 99% identical to the consensus sequence. Accordingly,polypeptides of the invention are about 50%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to theconsensus sequence.

A “consensus amino acid” is an amino acid chosen to occupy a givenposition in the consensus protein. A system which is organized to selectconsensus amino acids can be a computer program, or a combination of oneor more computer programs with “by hand” analysis and calculation. Whena consensus amino acid is obtained for each position of the alignedamino acid sequences, then these consensus amino acids are “lined up” toobtain the amino acid sequence of the consensus protein.

As mentioned above, modification and changes may be made in thestructure of the mucE coding region and still obtain a molecule havinglike or otherwise desirable characteristics. As used herein, the term“biological functional equivalent” refers to such proteins. For example,certain amino acids may be substituted for other amino acids in aprotein structure without appreciable loss of interactive bindingcapacity with structures such as, for example, antigen-binding regionsof antibodies or binding sites on substrate molecules. Since it is theinteractive capacity and nature of a protein that defines that protein'sbiological functional activity, certain amino acid sequencesubstitutions can be made in the DNA coding sequence and neverthelessobtain a protein with like or even counterveiling properties (e.g.,antagonistic v. agonistic). It is thus contemplated by the inventorsthat various changes may be made in the DNA sequence of mucE (or MucEproteins or peptides) without appreciable loss of their biologicalutility or activity.

Polypeptides

Further embodiments of the invention include polypeptides, whichcomprise amino acid sequences at least 95% identical, and morepreferably at least 96%, 97%, 98% or 99% identical, to any of the aminoacid sequences of the polypeptides described above.

As a practical matter, whether any particular polypeptide is at least95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acidsequence shown in SEQ ID NOs:2 and 4 can be determined conventionallyusing known computer programs such as the Bestfit program (WisconsinSequence Analysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park 575 Science Drive, Madison, Wis. 53711).Bestfit uses the local homology algorithm of Smith and Waterman,Advances in Applied Mathematics 2:482-489 (1981), to find the bestsegment of homology between two sequences. When using Bestfit or anyother sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set, of course,such that the percentage of identity is calculated over the full lengthof the reference amino acid sequence and that gaps in homology of up to5% of the total number of amino acids in the reference sequence areallowed.

Even though the invention has been described with a certain degree ofparticularity, it is evident that many alternatives, modifications, andvariations will be apparent to those skilled in the art in light of theforegoing disclosure. Accordingly, it is intended that all suchalternatives, modifications, and variations which fall within the spiritand the scope of the invention be embraced by the defined claims.

The following examples are included for purposes of illustration onlyand are not intended to limit the scope of the present invention, whichis defined by the appended claims. It should be appreciated by those ofskill in the art that the techniques disclosed in the examples whichfollow, represent techniques discovered by the inventors to functionwell in the practice of the invention, and thus can be considered toconstitute preferred modes for its practice. However, those of skill inthe art should, in light of the present disclosure, appreciate that manychanges can be made in the specific embodiments which are disclosed andstill obtain a like or similar result without departing from the spiritand scope of the invention.

Antibodies

Further embodiments of the invention include MucE and AlgW monoclonalantibodies and methods of using MucE and AlgW antibodies for thedetection and diagnosis of mucoid P. aeruginosa in biological specimens.The methods comprise reacting a specimen suspected of containing mucoidP. aeruginosa with a MucE or AlgW monoclonal antibody or fragmentthereof, separating the specimen from unbound antibody, and detectingthe presence of immune complexes formed between the monoclonal antibodyand the MucE or AlgW protein, as compared to non-mucoid control cellsand therefrom determining the presence of mucoid P. aeruginosa. Novelhybrid cell lines are also provided which produce the monoclonalantibodies capable of specifically binding to the MucE or AlgW proteinexpressed in P. aeruginosa. When the monoclonal antibodies are labeledand combined with a solubilizing reagent, a specific and rapid directtest for mucoid P. aeruginosa is achieved.

The monoclonal antibodies of this invention can be prepared byimmortalizing the expression of nucleic acid sequences which code forantibodies specific for MucE or AlgW of P. aeruginosa. This may beaccomplished by introducing such sequences, typically cDNA encoding forthe antibody, into a host capable of cultivation and culture. Theimmortalized cell line may be a mammalian cell line that has beentransformed through oncogenesis, by transfection, mutation, or the like.Such cells include myeloma lines, lymphoma lines, or other cell linescapable of supporting the expression and secretion of the antibody invitro. The antibody may be a naturally occurring immunoglobulin of amammal other than human, produced by transformation of a lymphocyte, bymeans of a virus or by fusion of the lymphocyte with a neoplastic cell,e.g., a myeloma, to produce a hybrid cell line. Typically, the lymphoidcell will be obtained from an animal immunized against MucE or afragment thereof containing an epitopic site.

Monoclonal antibody technology was pioneered by the work of Kohler andMilstein, Nature 256:495 (1975). Monoclonal antibodies can now beproduced in virtually unlimited quantities consistently and with a highdegree of purity. These qualities facilitate the reproducibility andstandardization of performance of diagnostic tests which are required inhospitals and other clinical settings.

Immunization protocols are well known and can vary considerably yetremain effective. See Golding, Monoclonal Antibodies: Principles andPractice, (1983) which is incorporated herein by reference Immunogenicamounts of antigenic MucE preparations are injected, generally atconcentrations in the range of 1 ug to 20 mg/kg of host. Administrationof the antigenic preparations may be one or a plurality of times,usually at one to four week intervals Immunized animals are monitoredfor production of antibody to the desired antigens, the spleens are thenremoved and splenic B lymphocytes isolated and transformed or fused witha myeloma cell line. The transformation or fusion can be carried out inconventional ways, the fusion technique being described in an extensivenumber of patents, e.g., U.S. Pat. Nos. 4,172,124; 4,350,683; 4,363,799;4,381,292; and 4,423,147. See also Kennett et al., Monoclonal Antibodies(1980) and references therein.

The biological sample suspected of containing P. aeruginosa is combinedwith the primary antibody under conditions conducive to immune complexformation. If the test is a one-step immunofluorescence assay, theprimary antibody will be labeled. Typically, the specimen is first fixedor adhered to a glass slide by heat and/or ethanol treatment, althoughother fixatives or adherents are known by those skilled in the art. Thespecimen is then contacted with the solubilizing agent for a sufficientperiod, usually from 1 to 30 minutes and more usually about 10 minutes,and the solubilizer is then washed from the slide. Alternatively, asdescribed above, the solubilizing agent and the primary antibody may becombined and added as one step. The primary antibody should be incubatedwith the specimen for approximately 30 minutes at room temperature,although the conditions may be varied somewhat. The slide is rinsed toremove unbound antibody. If the primary antibody has been labeled withFITC, the reacted sample may be viewed under a fluorescence microscopeequipped with standard fluorescein filters (excitation=490 nm;emission=520 nm) and a 40× oil immersion lens. The quantitation offluorescence is based on visual observation of the brightness orrelative contrast of the specifically stained antigen. Appropriatepositive and negative controls make interpretation more accurate. Acounterstain, such as Evans blue, may be employed to more easilyvisualize the fluorescent organisms.

The antibodies of the invention may be a chimeric antibody or fragmentthereof, a humanized antibody or fragment thereof, a single chainantibody; or a Fab fragment.

For use in diagnostic assays, the antibodies of the present inventionmay be directly labeled. A wide variety of labels may be employed, suchas radionuclides, fluorescence, enzymes, enzyme substrates, enzymecofactors, enzyme inhibitors, ligands (particularly haptens), etc. Whenunlabeled, the antibodies may find use in agglutination assays. Inaddition, unlabeled antibodies can be used in combination with otherlabeled antibodies (second antibodies) that are reactive with themonoclonal antibody, such as antibodies specific for the immunoglobulin.Numerous types of immunoassays are available and are well known to thoseskilled in the art.

Immunofluorescence staining methods can be divided into two categories,direct and indirect. In the direct staining method, a fluorophore isconjugated to an antibody (the “primary antibody”) which is capable ofbinding directly to the cellular antigen of interest. In the indirectstaining mode, the primary antibody is not fluorescently labeled;rather, its binding is visualized by the binding of a fluorescentlylabeled second-step antibody, which second-step antibody is capable ofbinding to the primary antibody. Typically, the second-step antibody isan anti-immunoglobulin antibody. In some instances the second-stepantibody is unlabeled and a third-step antibody which is capable ofbinding the second-step antibody is fluorescently labeled.

Indirect immunofluorescence is sometimes advantageous in that it can bemore sensitive than direct immunofluorescence because for each moleculeof the primary antibody which is bound, several molecules of the labeledsecond-step antibody can bind. However, it is well known that indirectimmunofluorescence is more prone to nonspecific staining than directimmunofluorescence, that is, staining which is not due to the specificantigen-antibody interaction of interest (Johnson et al., in Handbook ofExperimental Immunology, D. M. Weir, ed., Blackwell Publications Oxford(1979); and Selected Methods in Cellular Immunology, Mishell et al.,ed., W. H. Freeman, San Francisco (1980)). In addition, the multiplesteps involved in performing the indirect tests makes them slow, laborintensive, and more susceptible to technician error.

Various immunoassays known in the art can be used to detect binding ofMucE or AlgW to antibodies, including but not limited to, competitiveand non-competitive assay systems using techniques such asradioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoradiometric assays, gel diffusion precipitationreactions, immunodiffusion assays, in situ immunoassays (using colloidalgold, enzyme or radioisotope labels, for example), western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc.

Kits can also be supplied for use with the subject antibodies in thedetection of mucoid P. aeruginosa in specimens, wherein the kitscomprise compartments containing a MucE and/or AlgW monoclonal antibodycapable of reacting with essentially all serotypes and immunotypes of P.aeruginosa, and labels and necessary reagents for providing a detectablesignal. Thus, the monoclonal antibody composition of the presentinvention may be provided, usually in a lyophilized form, either aloneor in conjunction with additional antibodies specific for other antigensof P. aeruginosa. The antibodies, which may be conjugated to a label,are included in the kits with buffers, such as Tris, phosphate,carbonate, etc., stabilizers, biocides, inert proteins, e.g., bovineserum albumin, or the like. Generally, these materials will be presentin less than about 5% weight based on the amount of active antibody, andusually present in a total amount of at least about 0.001% weight basedon the antibody concentration. Frequently, it will be desirable toinclude an inert extender or excipient to dilute the active ingredients,where the excipient may be present in from about 1% to 99% weight of thetotal composition. Where a second antibody capable of binding to themonoclonal antibody is employed, this will usually be present in aseparate vial. The second antibody may be conjugated to a label andformulated in a manner analogous to the antibody formulations describedabove.

Cystic Fibrosis (CF) Risk Assessment

Further embodiments of the invention include methods for Cystic Fibrosis(CF) disease assessment in an individual which comprise detecting thepresence or absence of MucE and/or AlgW in a sample from an individual.Further embodiments include methods for Cystic Fibrosis (CF) diseaseassessment in an individual which comprise detecting the presence orabsence of MucE or AlgW antibodies in a sample from an individual.

Additional embodiments include methods for treating P. aeruginosabiofilms in Cystic Fibrosis (CF) disease in an individual which comprisethe steps of detecting the presence of MucE and/or AlgW in a sample froman individual; and selecting a therapy regimen for the individual basedon the presence of MucE and/or AlgW. The P. aeruginosa biofilms inCystic Fibrosis (CF) disease are treated by the therapy regimen. Alsocontemplated are methods for treating P. aeruginosa biofilms in CysticFibrosis (CF) disease in an individual which comprise the steps ofdetecting the presence of MucE and/or AlgW antibodies in a sample froman individual; and selecting a therapy regimen for the individual basedon the presence of MucE and/or AlgW antibodies. The P. aeruginosabiofilms in Cystic Fibrosis (CF) disease are treated by the therapyregimen.

As used herein, “individual” is intended to refer to a human, includingbut not limited to, children and adults. One skilled in the art willrecognize the various biological samples available for detecting thepresence or absence of MucE or AlgW in an individual, any of which maybe used herein. Samples include, but are not limited to, airway surfaceliquid, sputa, or combinations thereof, human blood, wound exudate,respiratory secretions, human tissues (e.g., lung) or a laboratoryculture thereof, and urine. Moreover, one skilled in the art willrecognize the various samples available for detecting the presence orabsence of MucE or AlgW antibodies in an individual, any of which may beused herein. Samples include, but are not limited to, airway surfaceliquid, sputa, or combinations thereof, human blood, wound exudate,respiratory secretions, human tissues (e.g., lung) or a laboratoryculture thereof, urine, and other body fluids, or combinations thereof.

As used herein, “assessment” is intended to refer to the prognosis,monitoring, delaying progression, delaying early death, staging,predicting progression, predicting response to therapy regimen,tailoring response to a therapy regimen, of Cystic Fibrosis diseasebased upon the presence or absence of MucE, AlgW, MucE antibodies, orAlgW antibodies in a biological sample.

As used herein, “therapy regimen” is intended to refer to a procedurefor delaying progression, or delaying early death associated with CysticFibrosis disease and/or Pseudomonas aeruginosa in a Cystic Fibrosisindividual. In one embodiment, the therapy regimen comprisesadministration of agonists and/or antagonists of MucE and/or AlgW. Inanother embodiment, the therapy regimen comprises agonists and/orantagonists of Pseudomonas aeruginosa.

One skilled in the art will appreciate the various known direct and/orindirect techniques for detecting the presence or absence of MucE orAlgW, any of which may be used herein. These techniques include, but arenot limited to, amino acid sequencing, antibodies, Western blots,2-dimensional gel electrophoresis, immunohistochemistry,autoradiography, or combinations thereof.

All references cited in the Examples are incorporated herein byreference in their entireties.

EXAMPLES

Materials and Methods

The following materials and methods apply generally to all the examplesdisclosed herein. Specific materials and methods are disclosed in eachexample, as necessary.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology (including PCR), vaccinology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See, for example,Molecular Cloning A Laboratory Manual, 2d Ed., Sambrook et al., ed.,Cold Spring Harbor Laboratory Press (1989); DNA Cloning, Volumes I andII, D. N. Glover ed., (1985); Oligonucleotide Synthesis, M. J. Gait ed.,(1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic AcidHybridization, B. D. Hames & S. J. Higgins eds. (1984); TranscriptionAnd Translation, B. D. Hames & S. J. Higgins, eds. (1984); Freshney, R.I., Culture Of Animal Cells, Alan R. Liss, Inc. (1987); ImmobilizedCells And Enzymes, IRL Press (1986); Perbal, B., A Practical Guide ToMolecular Cloning (1984); the treatise, Methods In Enzymology, AcademicPress, Inc., N.Y.; Gene Transfer Vectors For Mammalian Cells, J. H.Miller and M. P. Calos eds., Cold Spring Harbor Laboratory (1987);Methods In Enzymology, Vols. 154 and 155, Wu et al. eds.; ImmunochemicalMethods In Cell And Molecular Biology, Mayer and Walker, eds., AcademicPress, London, (1987); and in Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1989). Each ofthe references cited in this paragraph is incorporated herein byreference in its entirety.

Bacterial Strains, Plasmids, Transposons and Growth Conditions

P. aeruginosa strains were grown at 37° C. in Lennox broth (LB), on LBagar or Pseudomonas Isolation Agar (PIA, DIFCO) plates. When required,PIA plates were supplemented with carbenicillin, tetracycline, orgentamicin at a concentration of 300 ng/ml. E. coli strains were grownin LB broth, or LB agar supplemented with carbenicillin (100 μg/ml),tetracycline (15 μg/ml), gentamicin (13 μg/ml), or kanamycin (40 μg/ml),when required.

Transposon Mutagenesis

A standard Pseudomonas conjugation protocol was followed with thefollowing modifications. E. coli SM10 λpir carrying pFAC and P.aeruginosa strains were grown in 2 ml LB broth overnight at 37° C. and42° C., respectively. The cell density of the cultures was measured byoptical density at 600 nm and adjusted to a ratio of 1:1, which wasequivalent to 8×10⁸ cells for matings. The mixed cultures were incubatedon LB plates for 6 h at 37° C. The cells were harvested and washed in LBbroth. The final cell mixtures in a volume of 1 ml were spread on 8 PIAplates (50 ml each) supplemented with gentamicin. The conjugal pairswere incubated at 37° C. for 24 h for selection and screeningexconjugants with a mucoid colony morphology. Such mutants were isolatedand purified a minimum of 3 times. Mutants were frozen in 10% skim milkin a −80° C. freezer.

DNA Manipulations.

Two steps of polymerase chain reaction (PCR)-based cloning were used forgeneral cloning purposes. First, the target genes were amplified byhigh-fidelity PCR using the appropriate primer sets containing thebuilt-in restriction sites followed by cloning into pCR4-TOPO. The DNAfragments were digested by restriction enzymes, gel-purified, andtransferred to the shuttle vector pUCP20. All recombinant plasmids weresequenced to verify the absence of mutations with M13 universal forwardand reverse primers using an ABI 3130 Genetic Analyzer at the MarshallUniversity School of Medicine Genomics Core Facility. PCR reactions wereperformed with MasterAmp™ Taq DNA Polymerase (Epicentre) in 50 μlEasyStart PCR tubes (Molecular BioProducts) as previously described(Head, N. E., and H. Yu, Infect. Immun. 72:133-44 (2004)).

Inverse PCR (iPCR)

The mariner transposon and its junction region in pFAC were sequenced.The sequence of the junction region including the inverted repeats inpFAC (SEQ ID NO:8) is as follows:

accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc actcaaccaagtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacgggataataccgcg ccacataaca ggttggctga taagtccccg gtctaacaaa gaaaaacacatttttttgtg aaaattcgtt tttattattc aacatagttc ccttcaagag cgatacccctcgaattgacg cgtcaattct cgaattgaca taagcctgtt cggttcgtaa actgtaatgcaagtagcgta tgcgctcacg caactggtcc agaaccttga ccgaacgcag cggtggtaacggcgcagtgg cggttttcat ggcttgttat gactgttttt ttgtacagtc tatgcctcgggcatccaagc agcaagcgcg ttacgccgtg ggtcgatgtt tgatgttatg gagcagcaacgatgttacgc agcagcaacg atgttacgca gcagggcagt cgccctaaaa caaagttaggtggctcaagt atgggcatca ttcgcacatg taggctcggc cctgaccaag tcaaatccatgcgggctgct cttgatcttt tcggtcgtga gttcggagac gtagccacct actcccaacatcagccggac tccgattacc tcgggaactt gctccgtagt aagacattca tcgcgcttgctgccttcgac caagaagcgg ttgttggcgc tctcgcggct tacgttctgc ccaggtttgagcagccgcgt agtgagatct atatctatga tctcgcagtc tccggcgagc accggaggcagggcattgcc accgcgctca tcaatctcct caagcatgag gccaacgcgc ttggtgcttatgtgatctac gtgcaagcag attacggtga cgatcccgca gtggctctct atacaaagttgggcatacgg gaagaagtga tgcactttga tatcgaccca agtaccgcca cctaacaattcgttcaagcc gagatcggct tcccggccga cgcgtcctcg gtaccgggcc ccccctcgaggtcgacggta tcgataagct tgatatcgaa ttcctgcagc ccgggaatca tttgaaggttggtactatat aaaaataata tgcatttaat actagcgacg ccatctatgt gtcagaccggggacttatca gccaacctgt tagcagaact ttaaaagtgc tcatcattgg aaaaaggctgcgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaagggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgttgtaaaacga cggccagtga gcgcgcgtaa tacactcact atagggcgaa ttggaggatccggtctaaca aagaaaacac attttttgtg aaa

A multiple cloning site (MCS) was identified immediately outside the 3′end of the gentamicin cassette within the transposon. To map theinsertion site, an iPCR protocol was developed to utilize thisconvenient MCS. Pseudomonas genomic DNA was purified using a QIAampgenomic DNA kit. The DNA concentration was measured using the NanoDrop®ND-1000 spectrophotometer (NanoDrop Technologies). Two μg DNA wasdigested by restriction enzymes SalI or PstI at 37° C. overnightfollowed by gel purification. The fragmented DNA was ligated to form thecircularly closed DNA using the Fast-Link™ DNA ligation kit (Epicentre).A volume of 1 μl ligated DNA was used as template for PCR using GM5OUTand GM3OUT according to the condition as follows, 94° C. for 1 min, 34cycles consisting of 94° C. for 1 min, 58° C. for 2 min, and 72° C. for2 min, and a final extension step consisting of 72° C. for 8 min. AfterPCR, the products were analyzed on a 1% agarose gel. The PCR productswere purified using the QIAquick PCR purification kits and sequencedusing GM5OUT as described above.

Alginate and Protein Assays

The alginate assay was based on a previously published method (Knutson,C. A., and A. Jeanes, Anal. Biochem. 24:470-481 (1968)) with thefollowing modifications. P. aeruginosa and mutants were grown on 50 mlPIA plates in triplicate for a period of 72 h. At various time points,bacterial growth was removed from plates and re-suspended in 40 mlphosphate-buffered saline (PBS; pH 7.4). The optical density at 600 nm(OD₆₀₀) was recorded. The alginate standard curve was made usingD-mannuronic acid lactone (Sigma) in the range of 0-100 μg/ml. Tomeasure the protein concentration, the cells in PBS were lysed in 1:1ratio with 1M NaOH for 15 min. The protein assay was performed using theBio-Rad D_(c) Protein Assay kit. The range for protein standard (bovineserum albumin) curve was from 0.2 to 1.2 mg/ml.

β-Galactosidase Activity Assay

The assay was based on the method as originally described by Miller (InExperiments in Molecular Genetics, J. H. Miller, ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1972), pp. 352-355) with thefollowing modification. The cells of NH1-3 were grown on PIA plates intriplicate for 24 h at 37° C. The cells were harvested in PBS and celldensity was measured by OD₆₀₀. Samples were assayed after SDS/chloroformpermeabilization of the cells.

Alkaline Phosphatase A-Fusion Assay

The entire open reading frame and different portion of mucE weretranslationally fused with the E. coli phoA gene with deletion of thesequence encoding the N-terminal signal sequence. These mucE-phoAfusions were cloned into pUCP20 vector for alkaline phosphatase A-fusionassay as previously described (Lewenza, S. et al., Genome Res.15:321-329 (2005); Manoil, C. et al., J. Bacterol. 172:515-518 (1990))and the transformants were plated on the LB plate containing 40 μg/mlBCIP. The construct pUCP20-phoA expressing full-length PhoA was used asa positive control and the pUCP20-phoA expressing the truncated PhoAwithout N-terminal signal leader sequence as a negative control.

RNA Isolation and RT-PCR

P. aeruginosa strains PAO1, VE2 and VE3 were grown on 50 ml PIA platesfor 24 h at 37° C. The cells were harvested in 40 ml PBS andre-suspended based on OD₆₀₀ to produce a cell population of 10⁹ to 10¹⁰.Total RNA was isolated using a RiboPure™-Bacteria Kit (Ambion) followedby DNase treatment as supplied. The quality of RNA was evaluated on anAgilent 2100 bioanalyzer. RT-PCR was performed using a One-Step RT-PCRkit (Qiagen). One μg bacterial RNA was reverse-transcribed into cDNA at50° C. for 30 min followed by PCR amplification: 94° C. for 15 min, 34cycles consisting of 94° C. for 1 min, 58° C. for 2 min, and 72° C. for2 min. The PCR products were analyzed on 1% agarose gel, and theintensity of bands was analyzed on a Typhoon 8600 Variable Mode Imager(Molecular Dynamics) with the ImageQuant (v. 5.2) Software.

Monoclonal Antibodies

The AlgU and MucB monoclonal antibodies used in the Examples are frompreviously published sources (Boucher et al., J. Bacteriol. 178:511-523(1996); Schurr et al., J. Bacteriol. 178:4997-5004 (1996)) with a lowlevel of cross-reactivity. The specificities of these antibodies areappropriate because the algU and mucB negative strains failed to displaythe respective AlgU and MucB proteins (FIG. 8). Furthermore, twonon-specific proteins of 50 kDa and 75 kDa from MucB and AlgU blotsrespectively were used as convenient internal controls to normalize theprotein levels.

Southern Hybridization

A 754 by PCR product was amplified from acc1 of pUCP30T using GM-F andGM-R primers, which was purified via gel extraction and labeled withdigoxygenin as described by the manufacturer (Roche MolecularBiochemicals). Agarose gels were soaked in 0.25 N HCl for 30 min, rinsedin H₂O, soaked in 1.5 M NaCl/0.5 M NaOH for 30 min and 1.5 M NaCl/0.5 MTris-Cl, pH 8.0 for 30 min. A blotting apparatus (BIO-RAD VacuumBlotter) was used with a filter paper wick, a Hybond-N+ membrane(Amersham Pharmacia Biotech), and transferred with 10×SSC transferbuffer for 2 h. After transfer, the membrane was rinsed in transferbuffer and UV cross-linked. Hybridization was done using the DIG HighPrime DNA Labeling and Detection Starter Kit II (Roche Applied Science)and labeled probe described above.

Western Blot Analysis.

Forty μg of total protein was prepared by bead-beating 3× for 1 min with5 min intervals on ice. The proteins were mixed with 2×SDS-PAGE samplebuffer. A Precision Plus Protein Standard (Bio-Rad) was used asmolecular mass ranging from 10 to 250 kD. Protein and standard wereloaded into a Criterion pre-cast gel of linear gradient (10-15% Tris-HClgel) (Bio-Rad) and was run in a Criterion Cell (Bio-Rad) at 60V for 4 h.The transfer onto a PVDF membrane was done in a Criterion Blotter(Bio-Rad) with CAPS buffer at 50V for 1 h. Primary antibodies wereobtained using standard techniques. Horseradish Peroxidase-labeledsecondary antibodies, goat anti-mouse IgG (H+L) and goat anti-rabbit IgG(H+L), were obtained from Peirce Biotechnologies and Kirkegaard & PerryLaboratories, respectively. Primary antibodies were diluted 1:1000 andsecondary antibodies 1:5000 in TBS/Tween before application. ECL WesternBlotting Detection System (Amersham Biosciences) was used to detect theprotein of interest. X-ray film was exposed, and developed on anAlphatek AX390SE developer. The protein intensity was analyzed using aChemiDoc XRS system (Bio-Rad) and Quantity One software (Bio-Rad). Theseresults were normalized against an internal protein within each sample.The relative expression level for each protein was then compared.

Statistical Analysis

Analysis of alginate production β-galactosidase activity was done withone-way analysis of variance (ANOVA) followed by pairwise multiplecomparisons with Holm-Sidak method. Analysis of normalized proteinintensity was carried out with the means of each group in comparisonwith that of PAO1 using t test assuming unequal variance or ANOVA ifmultiple groups were compared. All analyses were performed withSigmaStat (v. 3.1, Systat Software) and SigmaPlot (v. 9.0, SystatSoftware) software.

Example 1 Mariner-Based Transposon Mutagenesis Approach to IdentifyMucoid Mutants in P. aeruginosa

To investigate alginate regulation in P. aeruginosa, the versatileTc1/mariner himar1 transposon carried on pFAC (GenBank Accession numberDQ366300), a Pseudomonas suicide plasmid, was used to mutagenize thenon-mucoid strains of P. aeruginosa coupled with a genetic screen formucoid mutants.

The transposition efficiency of this transposon is high and has beenshown to cause high-density insertions in P. aeruginosa (Wong, S. M. andMekalanos, J. J., Proc Natl Acad Sci US 97:10191-10196 (2000)).Moreover, this transposon can knockout, knockdown or induce expressionof the target gene depending on the nature of its insertion. The marinertransposon himar1 can jump onto the TA dinucleotides in non-essentialgenes. These sites are abundant in the genomes of P. aeruginosa strains.Based on the two completed genomes, there are 94,404 and 100,229 suchsites in PAO1 (Stover et al., Nature 406:959-964 (2000)) and PA14respectively, which gives rise to 17-18 per ORF. In addition, pFAC cancause increased or reduced expression of the target gene by insertinginto the intergenic region.

Four non-mucoid strains were subject to transposon mutagenesis. Onlythree regions were targeted in this background: i) 6× in the algUpromoter region, ii) 1× in mucA, and iii) 3× in the intergenic regionbetween algU and mucA (Table 1). The algU promoter mutants causedincreased expression of AlgU while the mucA and the algU-mucA intergenicmutants affected the activity of AlgU. These results indicate that AlgUhas a key role in alginate overproduction in PAO579NM.

A total of 370,000 clones were screened from 13 conjugations (Table 1).Eighty-five mucoid mutants were isolated with 90% carrying singleinsertions as verified by Southern blot analysis (data not shown). Tomap the site of transposon insertions, iPCR was performed with 90% ofPCR reactions producing single products. The iPCR results displayed a100% correlation with Southern blots. The iPCR products were used astemplates for DNA sequencing. Seventy-eight mutants with singleinsertions were mapped. We next created the criteria of differentiatingthe independent mutational events. Independent and non-sibling mutantswere defined as those carrying a transposon at different sites, or atthe same sites but were obtained through different matings. Using thesecriteria, a collection of 45 independent mucoid mutants was obtained andclassified in 9 different functional groups (Table 1). The mutagenesisapproach used here was at a saturating level because multiple insertionsat the same sites were repeatedly targeted (FIG. 5).

TABLE I Transposon mutagenesis analysis of alginate regulators in fournon-mucoid strains of P. aeruginosa PAO1 PAO579NM PA14 FRD2 Sum Freq #matings 4 3 3 3 13 # mutants screened 81,280 88,800 126,000 75,000371,080 # mucoid obtained 32 18 31 4 85 # independent mutants 21 10 11 345 mutation freq 3.9 × 10⁻⁴ 2.0 × 10⁻⁴ 2.5 × 10⁻⁴ 5.3 × 10⁻⁵Induction^(a) PA0762-algUpromoter  5 (23.8) 6 (60.0)  8 (72.7) 3 (100.0)22 49 PA4033-mucE 1 (4.8) 1 (9.1) 2 4 PA4082-cupB5 1 (4.8) 1 2Knockdown^(b) PA0762-algU 2 (9.5) 3 (30.0) 5 11 PA0973-oprL 1 (4.8) 1 2Knockout^(c) PA0763-mucA 1 (10.0) 1 2 PA0764-mucB 1 (4.8) 1 (9.1) 2 4PA0766-mucD  9 (42.9) 1 (9.1) 10 22 PA5484-kinB 1 (4.8) 1 2 ^(a,b,c)Thenumber of mutants obtained from each strain of P. aeruginosa is shown.Number inside a bracket denotes the percentage of a mutation in thetotal number of mutants within a strain.

Similar to another himar1 transposon vector of the same lineage butconstructed for M. tuberculosis studies (Rubin, E. J., et al., Proc NatlAcad Sci USA 96:1645-1650 (1999)), the transposon end in pFAC has notermination sequences. Therefore, three types of mutations can be causedby the transposon in this vector depending on how and where it isinserted on the genome. As shown in Table 1, when inserted in the algU,mucE or cupB5 promoter region, the transposon used its σ⁷⁰ promoter(P_(Gm)) (Wohlleben, W., et al., Mol Gen Genet. 217:202-208 (1989)) todirect the expression of the downstream genes. Reduced (knockdown)expression occurred when the transposon was inserted in the intergenicregion of algUmucA or immediately downstream of oprL with P_(Gm) in theopposite direction with regard to the upstream algU or oprL. When thetransposon was within the coding sequences, this produced stop codonsaway from insertion sites due to frameshift mutations, producing geneknockouts for mucA, mucB, mucD and kinB.

The mucoid phenotype poises a great demand for energy from the cells.The amount of alginate in mucoid mutants was initially lower than thatof the wild-type strain PAO1 (FIG. 6A-8A), suggesting that mucoidmutants may grow slower than the non-mucoid counterparts to compensatefor the energy demand.

Example 2 The Majority of Insertions are within algUmucABCD and Resultin Upregulation of AlgU

While all pFAC insertions were within five clusters (data not shown),the most frequent sites (49%) were in the algU promoters with thetransposons situated in the induction configuration. Since thealgUmucABC genes are co-transcribed (DeVries, C. A. & Ohman, D. E., JBacteriol 176:6677-6687 (1994); Firoved, A. M. & Deretic, V., JBacteriol 185:1071-1081 (2003)), the levels of AlgU and MucB weremeasured in these mutants. VE1, one of the representative promotermutants as shown in FIG. 5, was grown on PIA plates for quantificationof alginate and the protein levels of AlgU and MucB.

As the results show, compared to PAO1, VE1 produced increased amounts ofalginate from 24 to 72 h in concurrence with increased levels of AlgUand MucB (FIG. 6). The level of AlgU was higher than that of MucB(P=0.005). AlgU and MucB reached the steady-state level at 4 h andremained so for the rest of the time points. The algU mutants inPAO579NM, PA14 and FRD2 were mucoid and displayed the same trend as VE1regarding alginate production and protein levels of AlgU and MucB. Theseresults indicate that the algU promoter mutations were gain-of-functionand associated with an elevated level of AlgU.

Twenty eight percent of mucoid mutants had insertions in the codingregions of mucA, mucB and mucD (Table 1). The Alg⁺ phenotype of themucD⁻ mutants (DR8, VE19, VE14 23 and VE12) was complemented to Alg⁻ bymucD or mucBCD in trans. VE3 and V1, the equivalent of a triple knockoutof mucA⁻B⁻C⁻ in PAO1 and PAO579NM respectively, were complemented to Algby mucA, but not by mucBC or mucBCD, in trans. The Alg⁺ phenotype inmucB⁻ mutants of PAO1 (VE8) and PA14 (DR1) was complemented to Alg⁻ bymucB, mucBC and mucBCD, in trans. These results suggest that theinsertions in mucA, mucB and mucD are loss-of-function (null) mutations.

Example 3 mucE and cupB5 Encode Two Novel Positive Regulators ofAlginate

Alginate is regulated by a signal transduction pathway. While ampleinformation is available on the interaction between the sigma factorAlgU and trans-inner membrane anti-sigma factor MucA, it is unclear whatand how periplasmic signals activate the AlgU pathway leading toalginate overproduction. MucE and CupB5 identified here are twocandidates for such signals. VE2 and DR4 had two identical insertions 16bps upstream of ATG of PA4033 in PAO1 and PA14, respectively (data notshown). The transposon in both mutants was in the inductionconfiguration (Table 1). PA4033 belongs to a class of unclassified openreading frames (ORF) in the annotated genome of PAO1, and encodes ahypothetical peptide (89 aa) with a predicted molecular mass of 9.5 kDa.

The protein has a leader sequence of 36 aa with the mature MucE proteinexported to periplasm. In E. coli, the σ^(E) pathway is activated via asimilar signal transduction system in which an outer membrane porin,OmpC serves as an inducing signal. The carboxy-terminal signal of MucE(WVF) has a three consensus aa sequence as does OmpC (YQF) (Walsh, N.P., et al., Cell 113:61-71 (2003)) and CupB5 (NIW).

The results show that alginate production in VE2 was increased after 24h (FIG. 7A) in association with the increased levels of AlgU and MucBcompared with PAO1 at all time points (FIG. 7A vs. FIG. 6B). Thewild-type and mucoid mutation alleles of PA4033 plus its upstream regionwere cloned into pUCP20. The resultant plasmid was named pUCP20-Gm-MucE(5622 bp) and has the following nucleotide sequence (SEQ ID NO:9):

GACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGGATATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATAAGCTAGCTTATCGGCCAGCCTCGCAGAGCAGGATTCCCGTTGAGCACCGCCAGGTGCGAATAAGGGACAGTGAAGAAGGAACACCCGCTCGCGGGTGGGCCTACTTCACCTATCCTGCCCGGCTGACGCCGTTGGATACACCAAGGAAAGTCTACACGAACCCTTTGGCAAAATCCTGTATATCGTGCGAAAAAGGATGGATATACCGAAAAAATCGCTATAATGACCCCGAAGCAGGGTTATGCAGCGGAAAGTATACCTTAAGGAATCCCCATGTTCTTTCCTGCGTTATCCCCTGATTCTGCGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCACGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTATCACCAGGAAACAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCCTAGCTGATTCCAAATAGCCATTAAGCGGGACGAAGAGCCCGTGAGCCAGCGCCAGCCTGACCTAACAGGTTGGCTGATAAGTCCCCGGTCTAACAAAGAAAAACACATTTTTTTGTGAAAATTCGTTTTTATTATTCAACATAGTTCCCTTCAAGAGCGATACCCCTCGAATTGACGCGTCAATTCTCGAATTGACATAAGCCTGTTCGGTTCGTAAACTGTAATGCAAGTAGCGTATGCGCTCACGCAACTGGTCCAGAACCTTGACCGAACGCAGCGGTGGTAACGGCGCAGTGGCGGTTTTCATGGCTTGTTATGACTGTTTTTTTGTACAGTCTATGCCTCGGGCATCCAAGCAGCAAGCGCGTTACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGATGTTACGCAGCAGCAACGATGTTACGCAGCAGGGCAGTCGCCCTAAAACAAAGTTAGGTGGCTCAAGTATGGGCATCATTCGCACATGTAGGCTCGGCCCTGACCAAGTCAAATCCATGCGGGCTGCTCTTGATCTTTTCGGTCGTGAGTTCGGAGACGTAGCCACCTACTCCCAACATCAGCCGGACTCCGATTACCTCGGGAACTTGCTCCGTAGTAAGACATTCATCGCGCTTGCTGCCTTCGACCAAGAAGCGGTTGTTGGCGCTCTCGCGGCTTACGTTCTGCCCAGGTTTGAGCAGCCGCGTAGTGAGATCTATATCTATGATCTCGCAGTCTCCGGCGAGCACCGGAGGCAGGGCATTGCCACCGCGCTCATCAATCTCCTCAAGCATGAGGCCAACGCGCTTGGTGCTTATGTGATCTACGTGCAAGCAGATTACGGTGACGATCCCGCAGTGGCTCTCTATACAAAGTTGGGCATACGGGAAGAAGTGATGCACTTTGATATCGACCCAAGTACCGCCACCTAACAATTCGTTCAAGCCGAGATCGGCTTCCCGGCCGACGCGTCCTCGGTACCGGGCCCCCCCTCGAGGTCGACGGTATCGATAAGCTTGATATCGAATTCCTGCAGCCCGGGAATCATTTGAAGGTTGGTACTATATAAAAATAATATGCATTTAATACTAGCGACGCCATCTATGTGTCAGACCGGGGACTTATCAGCCAACCTGTTATCAAGGAGTCGTAGCCATGGGTTTCCGGCCAGTTAGCCAACGTTTGCGTGACATCAACCTGCAGGCCCTCGGCAAGTTTTCCTGCCTTGCCCTGGTCCTCGGCCTGGAATCGGTAAGCCATCCGGCCGGCCCGGTCCAGGCCCCCTCGTTCAGCCAGGGCACCGCCAGCCCGTCCTTCGCTACTCCGCTCGGCCTCGACGGCCCGGCCCGCGCCAGGGCCGAGATGTGGAACGTCGGCCTGTCCGGCGCCGTCAGCGTGCGTGACGAGTTGCGCTGGGTGTTTTGAACGCGAAGCTTAGGGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAAAGGCAGGCCGGGCCGTGGTGGCCACGGCCTCTAGGCCAGATCCAGCGGCATCTGGGTTAGTCGAGCGCGGGCCGCTTCCCATGTCTCACCAGGGCGAGCCTGTTTCGCGATCTCAGCATCTGAAATCTTCCCGGCCTTGCGCTTCGCTGGGGCCTTACCCACCGCCTTGGCGGGCTTCTTCGGTCCAAAACTGAACAACAGATGTGTGACCTTGCGCCCGGTCTTTCGCTGCGCCCACTCCACCTGTAGCGGGCTGTGCTCGTTGATCTGCGTCACGGCTGGATCAAGCACTCGCAACTTGAAGTCCTTGATCGAGGGATACCGGCCTTCCAGTTGAAACCACTTTCGCAGCTGGTCAATTTCTATTTCGCGCTGGCCGATGCTGTCCCATTGCATGAGCAGCTCGTAAAGCCTGATCGCGTGGGTGCTGTCCATCTTGGCCACGTCAGCCAAGGCGTATTTGGTGAACTGTTTGGTGAGTTCCGTCAGGTACGGCAGCATGTCTTTGGTGAACCTGAGTTCTACACGGCCCTCACCCTCCCGGTAGATGATTGTTTGCACCCAGCCGGTAATCATCACACTCGGTCTTTTCCCCTTGCCATTGGGCTCTTGGGTTAACCGGACTTCCCGCCGTTTCAGGCGCAGGGCCGCTTCTTTGAGCTGGTTGTAGGAAGATTCGATAGGGACACCCGCCATCGTCGCTATGTCCTCCGCCGTCACTGAATACATCACTTCATCGGTGACAGGCTCGCTCCTCTTCACCTGGCTAATACAGGCCAGAACGATCCGCTGTTCCTGAACACTGAGGCGATACGCGGCCTCGACCAGGGCATTGCTTTTGTAAACCATTGGGGGTGAGGCCACGTTCGACATTCCTTGTGTATAAGGGGACACTGTATCTGCGTCCCACAATACAACAAATCCGTCCCTTTACAACAACAAATCCGTCCCTTCTTAACAACAAATCCGTCCCTTAATGGCAACAAATCCGTCCCTTTTTAAACTCTACAGGCCACGGATTACGTGGCCTGTAGACGTCCTAAAAGGTTTAAAAGGGAAAAGGAAGAAAAGGGTGGAAACGCAAAAAACGCACCACTACGTGGCCCCGTTGGGGCCGCATTTGTGCCCCTGAAGGGGCGGGGGAGGCGTCTGGGCAATCCCCGTTTTACCAGTCCCCTATCGCCGCCTGAGAGGGCGCAGGAAGCGAGTAATCAGGGTATCGAGGCGGATTCACCCTTGGCGTCCAACCAGCGGCACCAGCGGCGCCTGAGAGGTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCG AAACGCGCGA

The PA4033 (VE2) but not wild-type PA4033 allele caused mucoidconversion in PAO1 and PA14 in association with an elevated level ofAlgU in comparison with the parent (data not shown). Because theinsertional mutation was dominant over the wild-type allele, wesuspected that VE2 was overproducing the mucE product. Using RT-PCR, wedetermined that the levels of mucE and algU mRNA were 7-fold and1.2-fold higher in VE2 (PAO1 mucE^(+oe)) than in PAO1 respectively (FIG.7B). Because of the positive effect on alginate regulation, PA4033 wasnamed mucE.

We also tested whether introduction of pUCP20-Gm^(r)-mucE orpUCP20-P_(Gm)-mucE plasmids could cause mucoid conversion in othernon-mucoid P. aeruginosa strains. We observed the emergence of a mucoidphenotype in the environmental isolate ERC-1 and non-mucoid clinical CFisolates including CF149 (Head, N. H. et al., Infect. Immun. 72:133-144(2004)) and early colonizing strains (C0746C, C0126C, C0686C, C1207C,C3715C, C4009C, C7406C and C8403C) (data not shown). Therefore, theextracytoplasmic stress signals may play an important role in theinitial lung colonization and mucoid conversion of P. aeruginosa.

Induction of MucE initiates a regulatory cascade causing an increasedlevel of AlgU. It appears that induction of AlgU is the major pathwaythat governs alginate overproduction. The mutants that operate via thispathway include VE1 (algU promoter mutants), VE2 (mucE^(+oe)), and VE22(cupB5^(+oe)), and VE13 (kinB⁻). One common feature is that an elevatedlevel of MucB did not seem to match with that of AlgU (FIGS. 6A-8A). Asthe algUmucA-D genes are an operon, this suggests that the level of MucAin these mutants may not be the same as that of AlgU. The excess AlgUcould escape from the antagonistic interaction with MucA, thus causingmucoid conversion.

Another mutant, VE22, which had a dominant effect on alginateoverproduction, carried an insertion at 96 by before ATG of cupB5(PA4082) (Table 1). The cupB5 gene encodes a probable adhesive protein(1,018 aa) with a predicted molecular mass of 100 kDa. This protein hasa signal peptide of 53 aa, suggesting that the mature protein is boundfor the extracellular milieu. The protein shares consensus motifs of thefilamentous hemagglutinin and IgA1-specific metalloendopeptidases (GLUG)at the N- and C-terminus, respectively. The cupB5 gene sits within agenetic cluster encoding fimbrial subunits and CupB5, which have beenproposed to be the chaperone/usher pathway involved in biofilm formation(Vallet, I., et al., Proc Natl Acad Sci USA 98:6911-6916 (2001)).Induction of cupB5 in VE22 caused upregulation of AlgU and MucB (FIG.8B).

Example 4 KinB is a Negative Regulator of Alginate in PAO1

As a sensor-kinase, KinB is responsible for responding to someenvironmental signals and phosphorylating a response regulator, AlgB,via signal transduction. One mutant, VE13, displayed a stable mucoidphenotype (Table 1). The mutation of VE13 was mapped to 788 bps afterATG of kinB. This insertion caused a frameshift mutation with a stopcodon created at 54 bps after the insertion site. To ensure thatinactivation of kinB was causal for the phenotype, PAO1 kinB was clonedinto pUCP20. Introduction of wild-type kinB in trans into VE13 reversedthe phenotype from Alg⁺ to Alg⁻. Alginate production in VE13 wassignificantly higher than that in PAO1, which was associated with theincreased amount of AlgU (P=0.005) while the level of MucB remainedunchanged (P=0.07) compared with PAO1 (FIGS. 8A and 6A).

The results show that the kinase activity inhibits overproduction ofalginate, thereby formally establishing the role of KinB as a negativeregulator of alginate. AlgB is a well-known transcriptional activatorfor alginate biosynthesis. VE13 is a kinB null mutant of PAO1, and theAlg⁺ phenotype has been complemented to Alg⁻ by pUCP-kinB in trans.Inactivation of kinB in PAO1 increased the levels of AlgU (FIG. 8A),suggesting that KinB may inhibit the expression of algU via anAlgB-independent fashion. Alternatively, since AlgB in VE13 is probablyin an un-phosphorylated or under-phosphorylated state, it is possiblethat this form of AlgB serves as the transcriptional activator foralginate.

Example 5 Reduced Expression of oprL Causes Mucoid Conversion in PAO1

One mutant, VE24, had an insertion at the stop codon (TAA) of oprL(PA0973) in the knockdown configuration. The oprL gene encodes a homologof the peptidoglycan associated lipoprotein precursor (168 aa) with apredicted molecular mass of 18 kDa. OprL has a leader sequence of 24 aawhich probably directs the mature protein to the outer membrane. Reducedexpression of oprL in VE24 caused mucoid conversion in PAO1, and wasassociated with a reduced level of AlgU and MucB (FIG. 8B).

Example 6 Nonmucoid Revertants in AlgU-Hyperactive Mutants were Causedby Suppressor Mutations Inactivating algU

Eleven percent of insertions were in the intergenic region between algUand mucA in the knockdown configuration (Table 1). The mutants of thiscategory were hyper mucoid. The level of AlgU in VE3 was slightlyreduced compared with that in PAO1 (FIG. 8B). The abundance of algU mRNAin VE3 was 84% of that in PAO1 based on RT-PCR (FIG. 7B). Four randomspontaneous non-mucoid revertants of VE3, PAO1-VE3-NM1-4, were isolated(GenBank accession numbers DQ352563, DQ352564, DQ352565, and DQ352566).Sequencing the algU gene in VE3-NM1, -NM2, -NM3 and -NM4 revealed thatall carried a completely inactivated algU gene due to tandemduplications or a nonsense mutation. The nucleotide sequences of thesefour algU mutants are:

VE3-NM1 (SEQ ID NO: 10): cgattcgctg ggacgctcga agctcctcca ggttcgaagaggagctttca tgctaaccca ggaacaggat cagcaactgg ttgaacgggt acagcgcggagacaagcggg ctttcgatct gctggtactg aaataccagc acaagatact gggattgatcgtgcggttcg tgcacgacgc ccaggaagcc caggacgtag cgcaggaagc cttcatcaaggcataccgtg cgctcggcaa tttccgcggc gatagtgctt tttatacctg gctgtatcggatcgccatca acaccgcgaa gaaccacctg gtcgctcgcg ggcgtcggcc accggacagcgatgtgaccg cagaggatgc ggagttcttc gagggcgacc acgccctgaa ggacatcgagtcgccggaac gggcgatgtt gcgggatgag atcgaggcca ccgtgcacca gaccatccagcagttgcccg aggatttgcg cacggccctg accctgcgcg agttcgaagg tttgagttacgaagatatcg ccaccgtgat gcagtgtccg gtggggacgg tgtccggtgg ggacggtacggtcgcggatc ttccgcgctc gtgaagcaat cgacaaagct ctgcagcctt tgttgcgagaagcctgacac agcggcaaat gccaagagag gtta VE3-NM2 (SEQ ID NO: 11):cttggcagac gattcgctgg gacgctcgaa gctcctccag gttcgaagag gagctttcatgctaacccag gaacaggatc agcaactggt tgaacgggta cagcgcggag acaagcgggctttcgatctg ctggtactga aataccagca caagatactg ggattgatcg tgcggttcgtgcacgacgcc caggaagccc aggacgtagc gcaggaagcc ttcatcaagg cataccgtgcgctcggcaat ttccgcggcg atagtgcttt ttatacctgg ctgtatcgga tcgccatcaacaccgcgaag aaccacctgg tcgctcgcgg gcgtcggcca ccggacagcg atgtgaccgcagaggatgcg gagttcttcg agggcgacca cgccctgaag gacatcgagt cgccggaacgggcgatgttg cgggatgaga tcgaggccac cgtgcaccag accatccagc agttgcccgaggatttgcgc acggccctga ccctctgcgc gagttcgaag gtttgagtta cgaagatatcgccaccgtga tgcagtgtcc ggtggggacg gtacggtcgc ggatcttccg cgctcgtgaagcaatcgaca aagctctgca gcctttgttg cgagaagcct gacacagcgg caaatgccaagagaggta VE3-NM3 (SEQ ID NO: 12): tatcttggca agacgattcg ctgggacgctcgaagctcct ccaggttcga agaggagctt tcatgctaac ccaggaacag gatcagcaactggttgaacg ggtacagcgc ggagacaagc gggctttcga tctgctggta ctgaaataccagcacaagat actgggattg atcgtgcggt tcgtgcacga cgcccaggaa gcccaggacgtagcgcagga agccttcatc aaggcatacc gtgcgctcgg caatttccgc ggcgatagtgctttttatac ctgactgtat cggatcgcca tcaacaccgc gaagaaccac ctggtcgctcgcgggcgtcg gccaccggac agcgatgtga ccgcagagga tgcggagttc ttcgagggcgaccacgccct gaaggacatc gagtcgccgg aacgggcgat gttgcgggat gagatcgaggccaccgtgca ccagaccatc cagcagttgc ccgaggattt gcgcacggcc ctgaccctgcgcgagttcga aggtttgagt tacgaagata tcgccaccgt gatgcagtgt ccggtggggacggtacggtc gcggatcttc cgcgctcgtg aagcaatcga caaagctctg cagcctttgttgcgagaagc ctgacacagc ggcaaatgcc aagagagta VE3-NM4 (SEQ ID NO: 13):gattcgctgg gacgctcgaa gctcctccag gttcgaagag gagctttcat gctaacccaggaacaggatc agcaactggt tgaacgggta cagcgcggag acaagcgggc tttcgatctgctggtactga aataccagca caagatactg ggattgatcg tgcggttcgt gcacgacgcccaggaagccc aggacgtagc gcaggaagcc ttcatcaagg cataccgtgc gctcggcaatttccgcggcg atagtgcttt ttatacctgg ctgtatcgga tcgccatcaa caccgcgaagaaccacctgg tcgctcgcgg gcgtcggcca ccggacagcg atgtgaccgc agaggatgcggagttcttcg agggcgacca cgccctgaag gacatcgagt cgccggaacg ggcgatgttgcgggatgaga tcgaggccac cgtgcaccag accatccagc agttgcccga ggatttgcgcacggccctga ccctgcgcga gttcgaaggt ttgagttacg aagatatcgc caccgtgatgcagtgtccgg tggggacggt gtccggtggg gacggtacgg tcgcggatct tccgcgctcgtgaagcaatc gacaaagctc tgcagccttt gttgcgagaa gcctgacaca gcggcaaatgccaagagagg ta

These mutations resulted in the disappearance of AlgU and MucB in thesemutants (FIG. 8B). The suppressor mutants were complemented to Alg⁺ byalgU in trans. The complemented mutants, which restored the mucoidphenotype, caused the re-appearance of AlgU (FIG. 8B). We also measuredthe AlgU-dependent P1 promoter activity by fusing the P_(algUP1) to thelacZ gene on the chromosome (DeVries, C. A. & Ohman, D. E., J Bacteriol176:6677-6687 (1994); Schurr, M. J., et al., J Bacteriol 176:3375-3382(1995)). Assay of the β-galactosidase activity indicated that theP_(algUP1) activity was 2348±156 units in NH1 (algU⁺) and 16.0±5.5 unitsin NH3 (algU⁻) while that of the promoterless control in NH2 was 146±34units (P=1.2×10⁻⁵) (data not shown).

PAO579 is a relatively unstable mucoid mutant of PAO1 origin with anundefined muc-23 mutation. A spontaneous non-mucoid revertant, PAO579NM,was isolated which had an unknown suppressor mutation. The algUmucAalleles in PAO579 and PAO579NM were sequenced but no mutations weredetected. To discern the pathway that regulated the mucoid phenotype inthis strain, PAO579NM was mutagenized to screen for mucoid mutants.Three sites, the algU promoter, the algUmucA intergenic region and mucA,were targeted that reversed the phenotype to Alg⁺ (Table 1). The highestfrequency of mutations (60% within the strain) occurred in the algUpromoters causing increased levels of AlgU and MucB in the same fashionas in VE1 in FIG. 5A (data not shown).

The results show that inactivation of mucA and mucB did not cause amarked induction in the amounts of AlgU and MucB to the same extent asthe kinB, mucE and cupB5 mutants (FIG. 8B vs. 6A-8A). This supports thenotion that the mucAB and oprL genes negatively regulate the activity ofAlgU (Firoved, A. M. & Deretic, V., J Bacteriol 185:1071-1081 (2003);Mathee, K., et al., J Bacteriol 179:3711-3720 (1997)).

Example 7 Upregulation of AlgU (AlgT) Causes Mucoid Conversion

The mucoid phenotype in clinical isolates of P. aeruginosa is unstable,and non-mucoid revertants arise spontaneously in the laboratory.Suppressor mutations in algT were the main cause of mucoid suppressionin P. aeruginosa (DeVries, C. A. & Ohman, D. E., J Bacteriol176:6677-6687 (1994); Schurr, M. J., et al., J Bacteriol 176:3375-3382(1994)). FRD2 is a CF isolate which has a suppressor mutation in algT18(DeVries, C. A. & Ohman, D. E., J Bacteriol 176:6677-6687 (1994)). Threerare mucoid mutants were identified in FRD2 (Table 1). They all had aninsertion in front of algU, in the same manner as the algU promotermutants in PAO1 (VE1), PA14, and PAO579NM, which resulted in increasedtranscription of the algT18mucA22mucBC operon as confirmed by Westernblots (FIG. 8B).

The rare FRD2 mucoid mutants coupled with the upregulation of AlgUsupport the notion that AlgU is the only sigma factor controlling theexpression of algD in P. aeruginosa (FIG. 8). The results indicate thata suppressor nonmucoid mutant (FRD2) can revert back to a mucoidphenotype (FRD2-VE1) in P. aeruginosa. This observation may help toexplain why the algU suppressors are prevalent in clinical isolates.

Analysis of the suppressor mutations in algU indicate that AlgU isrequired for alginate overproduction but is not an essential protein inP. aeruginosa.

Example 8 The Carboxyl Terminus of MucE Affects Mucoid Induction

The carboxyl-terminal signal of MucE (WVF) has a similar three consensusaa sequence as OmpC (YQF) (Walsh et al., 2003). Searching for this motifin the known outer membrane protein database from PAO1 did not identifyany obvious E. coli OmpC homologs, indicating that mucE encodes aprotein specific for induction of alginate. Other protein signals withsuch a function also exist. The C-terminal CupB5 carries the three aminoacid motif NIW. NIW and WVF are not interchangeable in MucE (unpublishedobservation), indicating that MucE and CupB5 work on different effectorproteins in the periplasm. Table II shows the effect of altering thecarboxyl terminus of MucE on mucoid induction in P. aeruginosa.

TABLE II Alteration of C-terminal signal moiety of MucE and mucoidyinduction in Pseudomonas aeruginosa PAO1. Carboxyl terminal MucoidyOuter membrane proteins with sequences induction the same C-terminalpeptide WVF M MucE (Wild-type) YVF M OprP, OprQ LVF M MucE orthologue(P. fluorences) WIF M MucE orthologue (P. syringae) WVW M WQF NM* YQFNM* OptS, HasR, OmpC and OmpF of E. coli. WLF NM DRF NM AlgE YYF NMStrongest signal in E. coli YKF NM OprH (PA1178) FQF NM AlgI WWW NM WVANM WVY NM ELR (ΔWVF) NM RWV (ΔF) NM M: mucoid.; NM: Non-mucoid;*Slightly mucoid after 1 day of incubation

The results in Table II show that the last three carboxyl-terminal aminoacids of MucE, WVF, are critical for the ability of MucE to inducemucoid induction.

Similarly, the WFV signal induced mucoidy in P. fluorescens. The WVF andYVF carboxyl terminal sequences significantly induced mucoidy, while theYQF carboxyl terminal sequence did not (data not shown). The envelopesignal is well conserved among Pseudomonads. Therefore, P. fluorescensis an alternative producer when alginate will be used for humanconsumption.

Example 9 MucE Interacts with AlgW Resulting in Alginate Overproduction

AlgW (GenBank accession number (U29172) is a periplasmic serine proteasein P. aeruginosa. Inactivation of algW on the chromosome of PAO1-VE2causes this strain to become nonmucoid (Boucher, J. C., et al., J.Bacteriol. 178:511-523 (1996)). Revertion back to the mucoid stateoccurs when a functional copy of algW is brought into the cells.Similarly, the disruption of algW in PAO1 (PA01ΔalgW) prevents mucoidinduction even when plasmid-borne mucE (pUCP20-Gm^(r)-mucE) was in astate of overexpression. MucE is found to interact with AlgW causingalginate overproduction by increasing the expression and/or activity ofAlgU.

Normally, AlgW is inactive because the functional domain (the trypsindomain) is covered with a PDZ domain of its own. Interaction betweenMucE and AlgW results in the release of the PDZ domain of AlgW. Thisinteraction occurs via the carboxyl terminus of MucE, specifically theterminal amino acids WVF, resulting in the activation of AlgW. ActivatedAlgW degrades the carboxyl terminus of anti-sigma factor MucA. Thisaction causes the release of AlgU into the cytoplasm, thereby activatingalginate biosynthesis (see FIG. 9). AlgU is the sigma factor that drivesalginate biosynthesis. Therefore, MucE is an inducing signal foralginate overproduction and the periplasmic target of MucE is AlgW (seeTable III).

TABLE III MucE-mediated induction of mucoidy in the mucA⁺ wild type P.aeruginosa is via AlgW. Bacterial strains Genotype Phenotype PAO1 Wildtype NM VE2 PAO1 over-expressing mucE M VE2 algW KO VE2 algW knockout NMVE2 algW KO + VE2 algW KO + M pUCP20algW pUCP20 algW

The nucleotide sequence of algW (SEQ ID NO:14) is as follows:

ATGCCCAAGGCCCTGCGTTTCCTCGGCTGGCCCGTGCTGGTCGGCGTGCTGCTGGCCCTGCTGATCATCCAGCACAACCCCGAGCTGGTCGGCCTGCCACGCCAGGAGGTGCACGTCGAGCAGGCGCCTCTGCTCAGCCGCCTGCAGGAAGGCCCGGTGTCCTATGCCAACGCGGTGAGTCGAGCGGCTCCGGCAGTGGCCAACCTGTACACCACCAAGATGGTCAGCAAGCCCTCCCACCCCCTGTTCGACGACCCGATGTTCCGCCGCTTCTTCGGCGACAACCTGCCGCAACAGAAGCGCATGGAGTCGAGCCTCGGCTCGGCGGTGATCATGAGCGCGGAAGGCTACCTGCTGACCAACAACCACGTGACCGCTGGCGCCGACCAGATCATCGTGGCCTTGCGCGACGGCCGCGAAACCATCGCCCAGTTGGTCGGCAGCGACCCGGAAACCGACCTGGCCGTGCTGAAGATCGACCTTAAGAACCTGCCGGCGATGACCCTCGGCCGCTCCGACGGCATTCGCACCGGCGACGTCTGCCTCGCCATCGGCAACCCGTTCGGCGTCGGCCAGACCGTGACCATGGGCATCATCAGCGCCACCGGACGCAACCAGCTCGGCCTGAACACCTACGAAGACTTCATCCAGACCGACGCGGCGATCAACCCCGGCAACTCCGGCGGCGCGCTGGTGGACGCTGCCGGCAACCTGATCGGCATCAACACGGCGATCTTCTCCAAGTCCGGCGGCTCCCAGGGTATCGGCTTCGCCATCCCGACCAAGCTGGCCCTGGAGGTCATGCAGTCGATCATCGAGCACGGCCAGGTGATCCGCGGCTGGCTCGGCGTCGAGGTCAAGGCGCTGACCCCGGAACTGGCGGAGTCGCTGGGCCTCGGCGAAACCGCCGGGATCGTCGTCGCCGGCGTCTATCGCGACGGTCCGGCGGCACGCGGCGGCCTGCTGCCGGGCGATGTGATCCTGACCATCGACAAGCAGGAAGCCAGCGACGGCCGCCGCTCGATGAACCAGGTGGCGCGCACCCGTCCGGGACAGAAGATCAGCATCGTGGTGCTGCGCAACGGACAGAAGGTCAACCTGACCGCCGAGGTCGGCCTGCGTCCGCCGCCGGCACCGGCTCCACAGCAGAAACAGGACGGCGGCGAGTGA

The amino acid sequence of AlgW (SEQ ID NO:15) is as follows:

MPKALRFLGWPVLVGVLLALLIIQHNPELVGLPRQEVHVEQAPLLSRLQEGPVSYANAVSRAAPAVANLYTTKMVSKPSHPLFDDPMFRRFFGDNLPQQKRMESSLGSAVIMSAEGYLLTNNHVTAGADQIIVALRDGRETIAQLVGSDPETDLAVLKIDLKNLPAMTLGRSDGIRTGDVCLAIGNPFGVGQTVTMGAIISTGRNQLGLNTYEDFIQTDAAINPGNSGGALVDAAGNLIGINTAIFSKQSGGSGIGFAIPTKLALEVMQSIIEHGQVIRGWLGVEVKALTPELAESLGLGETAGIVVAGVYRDGPAARGGLLPGDVILTIDKQEASDGRRSMNQVGARTRPQKISIVVLRNGQKVNLTAEVGLRPPPAPAPQQKQDGGE

The homolog of AlgW is DegS in E. coli (see also FIG. 12). Theinteraction between DegS and OmpC, an outer membrane porin protein, hasbeen shown to activate the signal transduction pathway for theactivation of RpoE, the AlgU homolog in E. coli. It has been shown thatinteraction between OmpC and DegS in the periplasm activates the signaltransduction pathway that controls the expression and/or activity ofRpoE, a homolog of AlgU (Walsh, N. P., et al., Cell 113:61-71 (2003)).

The results suggest that MucE functions upstream of the anti-sigmafactor MucA.

Example 10 The MucE Gene Encodes a Small Periplasmic or Outer MembraneProtein

The mucE gene is predicted to encode a polypeptide of 89 amino acidswith a probable transmembrane helix and a cleavable N-terminal signalsequence. (Stover, C. K., et al., Nature 406:959-964 (2000)). Homologuesof MucE are found in other species of pseudomonads capable of producingalginate (FIG. 11). We confirmed that mucE encodes a protein bydetecting an approximately 10 kD protein in Western blots of cellextracts of E. coli and P. aeruginosa expressing His-tagged MucE (FIG.13). PseudoCAP and Signal IP servers predicted that MucE is likely to belocated in the periplasm. To test the localization of MucE, weconstructed a series of deletions of mucE-phoA translational fusions. Weobserved phosphatase activity when phoA was fused to sequencecorresponding to the full-length MucE or the N-terminus after P36 butnot after A25. The MucE C-terminus-PhoA fusion did not show apparentphosphatase activity (FIG. 14). These results indicate that MucE is asmall protein of about 9.5 kDa located in the periplasm or outermembrane, with an N-terminal signal sequence that is required fortranslocation across the cytoplasmic membrane.

Example 11 MucP is Essential for MucE-Induced Conversion to Mucoidy

In E. coli, the degradation of RseA requires another protease calledRseP (also known as YaeL) to cleave the anti-sigma factor RseA after itis cleaved by DegS (Alba, B. M., et al., Genes Dev 16:2156-2168 (2002);Kanehara, K., et al., Embo J 22:6389-6398 (2003)). The P. aeruginosagenome also contains a homolog of RseP (PA3649, designated as MucP)(FIG. 15). The role of MucP in the degradation of MucA and activation ofAlgU activity was examined. Inactivation of mucP in PAO1VE2 caused aloss of mucoidy. Furthermore, the plasmid pUCP20 (pUCP20-mucP) restoredthe mucoid phenotype in PAO1VE2ΔmucP. Similarly, disruption of mucP inPAO1 prevented mucoid conversion when a high level of MucE was presentfrom plasmid pUC20-Gmr-mucE. In addition, a higher level of MucA and alower level of AlgU in PAO1VE2ΔmucP as compared to PAO1VE2 (data notshown) was seen. These results indicate that MucP is required for MucEactivation of AlgU activity.

Example 12 MucE-Induced Mucoidy does not Require the Prc Protease

The gene prc (PA3257) was recently identified as a regulator of alginatesynthesis in P. aeruginosa and is predicted to encode a PDZdomain-containing periplasmic protease similar to a E. coli proteasecalled Prc or Tsp (Reiling S. A., et al., Microbiology 151:2251-2261(2005)). Prc appears to act to promote mucoidy in mucA mutants bydegrading truncated forms of MucA found in mucoid mucA mutants (ReilingS. A., et al., Microbiology 151:2251-2261 (2005)). To test whether Prcplays a role in the activation of alginate production mediated by MucE,MucE was overexpressed in a strain lacking Prc and examined for mucoidy.Cells of the prc null mutant PAO1-184 (prc::tetR) carrying either MucEoverexpression plasmid pUCP20-Gmr-mucE or pUCP20-PGm-mucE were as mucoidas PAO1 cells carrying pUCP20-Gmr-mucE or pUCP20-PGm-mucE. These resultssuggest that Prc is not required for mucoidy induced by MucE and isconsistent with Prc only acting against truncated forms of MucA.

Example 13 MucD Eliminates Signal Proteins that Activate AlgW and OtherProteases to Cleave MucA

The mucD gene (PA0766) is a member of the algU mucABCD operon and ispredicted to encode a serine protease similar to HtrA in E. coli(Boucher, C. J., et al., J. Bacteriol. 178:511-523 (1996)). MucD appearsto be a negative regulator of mucoidy and AlgU activity (Boucher, C. J.,et al., J. Bacteriol. 178:511-523 (1996)). The mariner transposonlibrary screen confirmed this result because several mucoid mutants wereisolated that had transposons inserted within the coding region of mucD.HtrA in E. coli has been hypothesized to regulate the σ^(E) stressresponse system by removing misfolded proteins in the periplasm that canactivate the DegS protease via the degradation of the anti-sigma factorRseA (Alba, B. M., et al., Genes Dev. 16:2156-2168 (2002); Kanehara, K.,et al., Embo J. 22:6389-6398 (2003)). Therefore, it was determinedwhether MucD of P. aeruginosa acted in a similar manner as HtrA of E.coli. To test this, overexpression of MucD in a strain overexpressingMucE was examined Overexpression of mucD from the plasmid pUCP20-mucDpartially suppressed the mucoid phenotype of the mucE-overexpressingstrain PAO1VE2. This result is consistent with the notion that MucD canaid in the elimination of mis-folded OMPs including MucE. In addition,disruption of mucP in the mucoid mucD mutant PAO1VE19 caused the loss ofthe mucoid phenotype. The mucoid phenotype of PAO1VE19ΔmucP was restoredwhen mucP was in trans. Loss of the mucoid phenotype from the mucDmutant PAO1VE19 after the disruption of algW was not observed. Theresults suggest that MucD can act to remove misfolded proteins thatactivate proteases for degradation of MucA and that at least undercertain conditions other proteases independent of AlgW can also initiatethe cleavage of MucA.

1. An isolated polynucleotide, comprising a nucleic acid sequenceselected from the group consisting of SEQ ID NOS: 9-13.
 2. The isolatedpolynucleotide of claim 1, where the nucleic acid sequence comprises SEQID NO:
 9. 3. The isolated polynucleotide of claim 1, where the nucleicacid sequence comprises SEQ ID NO:
 10. 4. The isolated polynucleotide ofclaim 1, where the nucleic acid sequence comprises SEQ ID NO:
 11. 5. Theisolated polynucleotide of claim 1, where the nucleic acid sequencecomprises SEQ ID NO:
 12. 6. The isolated polynucleotide of claim 1,where the nucleic acid sequence comprises SEQ ID NO:
 13. 7. An isolatedpolynucleotide, comprising a nucleic acid sequence selected from SEQ IDNOS: 1 and
 3. 8. A isolated host cell, comprising the polynucleotide ofclaim
 7. 9. The isolated host cell of claim 8, wherein the host cell isa bacterial, yeast, animal, or plant cell.
 10. A vector, comprising theisolated polynucleotide of claim
 7. 11. The isolated polynucleotide ofclaim 7, wherein the isolated polynucleotide encodes the amino acidsequence of SEQ ID NO:
 2. 12. The isolated polynucleotide of claim 7,wherein the isolated polynucleotide encodes the amino acid sequence ofSEQ ID NO: 4.